Method of inducing a CTL response

ABSTRACT

Disclosed herein are methods for inducing an immunological CTL response to an antigen by sustained, regular delivery of the antigen to a mammal so that the antigen reaches the lymphatic system. Antigen is delivered at a level sufficient to induce an immunologic CTL response in a mammal and the level of the antigen in the mammal&#39;s lymphatic system is maintained over time sufficient to maintain the immunologic CTL response. Also disclosed is an article of manufacture for delivering an antigen that induces a CTL response in an animal.

CROSS REFERENCE

This application is a divisional of U.S. patent application Ser. No. 11/313,152 filed Dec. 19, 2005, now U.S. Pat. No. 7,364,729, which is a continuation of U.S. patent application Ser. No. 09/776,232, filed Feb. 2, 2001, now U.S. Pat. No. 6,977,074, which is a continuation-in-part of U.S. patent application Ser. No. 09/380,534, filed Sep. 1, 1999, now U.S. Pat. No. 6,994,851, which is a national stage entry of PCT Application No. PCT/US98/14289, filed Jul. 10, 1998, which is a continuation-in-part of U.S. patent application Ser. No. 08/988,320, filed Dec. 10, 1997, now abandoned, and which claimed priority to Canadian Patent Application No. 2,209,815, filed Jul. 10, 1997, each of which is hereby expressly incorporated by reference in its entirety.

SEQUENCE LISTING

The present application includes a Sequence Listing provided in electronic format on duplicate copies of a CD-ROM marked “Copy 1” and “Copy 2.” The duplicate copies of the CD-ROM each contain a file entitled MANNK.1CP2C1D2.txt created on Aug. 17, 2006 which is 88 kilobytes in size. The information on these duplicate CD-ROMs is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to a method of inducing a CTL response to an antigen by sustained, regular delivery of the antigen to an animal so that the antigen reaches the lymphatic system.

BACKGROUND OF THE INVENTION

Cytotoxic T lymphocytes (CTL) are white blood cells found in the blood, spleen and lymph. CTL have the ability to attack and kill other cells of the body in a highly specific manner. When CTL are stimulated by specific antigen, they migrate through the tissues of the body on a “search and destroy” mission for cells bearing the specific antigen. Whether of viral origin or tumor associated, CTL detect antigen that is bound to major histocompatability complexes (MHC) on the surface of potential target cells. Once CTL have identified the antigen on the cell surface, their function is to deliver a lethal hit to the cell.

Although there are hundreds of millions of CTL that reside in the spleen, each individual CTL exclusively responds to a unique and specific antigen. These individual CTL, dubbed CTL precursors (CTLp), undergo cell division or proliferate upon activation by specific antigen to produce daughter cells with precisely the same antigen specificity as the parent cell. This proliferation increases the total number, and thus the frequency, of that specific CTLp in the body. A proportion of these newly generated CTL briefly recirculate through the body (termed effector CTL), and have the ability to identify and destroy cells bearing the specific antigen which they recognize. A significant body of experimental evidence suggests that CTL specific for tumor antigens can inhibit tumor growth. Unfortunately, most tumors have only a very weak capacity to stimulate CTL responses and there has been no means of inducing a CTL response then sustaining it over a period of time sufficient to continuously inhibit tumor growth. While many attempts to directly increase the capacity of tumor cells to stimulate tumor-clearing CTL responses in patients have been made, such attempts have met with limited success. Technical advances over the past ten years have, however, enabled the identification of natural peptide antigens that are present on tumor cells and which are recognized by CTL. These antigen targets include proteins expressed in significant overabundance, abnormally expressed embryonic proteins, protein products from mutated oncogenes or suppressor genes, or proteins derived from cancer-causing viruses present in tumor cells. The challenge has been to find a way in which to administer an antigen so that it induces an antitumor CTL response and maintains it over time. While many attempts have now been made to use these antigens clinically in a vaccine, the results have been less than satisfactory.

An explanation of why CTL therapies have been largely ineffective at eradicating or controlling tumors in a clinical setting include the following:

(a) Vaccine designs have been inadequate at initiating strong CTL responses;

(b) Tumor cells can down regulate MI-IC molecules, resulting in the loss of antigen presentation from the surface of cells, thereby escaping detection by CTL;

(c) After induction, effector CTL recirculation through the body is highly transient;

(d) After recirculation, CTL return to the spleen where they reside in a nonactive or resting state, and an increase in the numbers of CTLp residing in the spleen does not reflect active CTL immunity;

(e) In the case of tumors, regrowth of residual tumor cells following immunization goes undetected by CTLp residing in spleen in a “resting” state;

(f) Because CTL-stimulating antigen presenting cells (APC are targeted for destruction by the same CTL that they have activated, the CTL response is self-limiting, which precludes, under normal circumstances, the continuous stimulation for a long-lived CTL response.

A growing repertoire of tumor associated antigens are being discovered that are recognized by CTL. A variety of techniques have been suggested to render these antigens effective in CTL vaccines. These include immunization using synthetic peptide antigens mixed with an immunostimulatory adjuvant, such as the bacterial toxin BCG; immunization with multiple antigenic peptide systems (MAPS), immunization with “professional” antigen presenting cells, which are isolated from the patient, pulsed with peptide antigen and inoculated back into the patient as a vaccine; immunization with peptides designed to stimulate both CTL and T helper cell populations; immunization with viruses or bacteria engineered to express tumor antigens; and immunization with polynucleotide expression vectors (so called DNA vaccines). Unfortunately, none of these approaches has been an unqualified success, As discussed above, the lack of vigorous therapeutic effects with these vaccine platforms reflects at least to some degree problems associated with inducing a strong initial CTL response and with maintaining ongoing “active” CTL immunity.

Studies by Glenny during the first quarter of the century revealed that aluminum compounds could enhance the strength of diphtheria vaccines. This was ostensibly the first of a long history of observations supporting a “depot” theory of immunization, which postulates that antigen slowly leaking into the tissues over an extended time correlates with the antigenic potency of a vaccine. Today, this antigen depot paradigm forms the intellectual backdrop to most adjuvant development programs. In one form or another, depot type adjuvants are intended to prolong the course of antigen delivery, by forming a lesion at the site of injection, or simply by the slow degradability of the adjuvant itself, which mixed with the specific antigen forms a depot at the site of injection. A second function generally attributed to adjuvants are their immunostimulatory effects, which appears to trigger the immune system to respond to the vaccine. However, adjuvants are a double-edged sword. They have inherent toxicities. But it is a feature of these toxicities that achieves a desired immunostimulatory and/or depot effect. Side effects such as tissue damage and granulornatous reaction at the site of injection, fever, and in some cases systemic reactions, such as Reiter's syndrome-like symptoms, uveitis and arthritis, are some of the risks associated with the use of adjuvants. Currently, the only adjuvant approved by the FDA is alum. It is relatively safe but does have side effects such as erythema, subcutaneous nodules, contact hypersensitivity, and granulornatous inflammation. More importantly, alum only acts to potentiate a limited number of antigens, and it very predominantly stimulates humoral antibody responses rather than CTL immunity. Thus so far adjuvants have proved to be very ineffective components for vaccines aimed at inducing clinically relevant CTL responses.

Recent attempts to induce CTL responses using dendritic cells or other antigen presenting cells, despite being cumbersome, have shown some promise. New recombinant virus or bacterial systems carrying genes for specific antigen are effective at inducing primary CTL responses. The most effective viruses, for example, that induce strong CTL responses are those which replicate aggressively in the host. Yet because of the risk for serious or lethal complications as a result of infection, recombinant virus used in a cancer vaccine must be only weakly replicative, or be completely replication deficient. This trade-off between virulence and efficacy is at present an intractable problem.

DNA (or polynucleotide) vaccines are also being developed for the purpose of inducing CTL immunity. Once again, the system has intrinsic limitations that preclude its efficacy in inducing long-lasting CTL immunity. The DNA vaccines consist of a plasmid or similar genetic construct for expressing the antigen of interest. Uptake of the plasmid system by cells of the body results in expression of the antigen and induction of CTL. However, once cells expressing the construct have succeeded in inducing CTL, they are themselves targets for eradication by the CTL. The CTL inducing effect is thus again transient. Moreover, the polynucleotide vaccines have thus far suffered from poor efficiency in terms of CTL induction.

With difficulties in achieving strong primary and/or persisting CTL responses, there are a number of clinical trial groups now using repeated injections of cancer vaccines. The use of antigenically complex materials in the vaccine formulation, such as recombinant virus, or the costs associated with repetitive treatment using cultured APC will, however, make such an approach difficult. On the one hand, repetitive immunization with antigenically complex materials drives the immune system to elaborate a humoral antibody, as opposed to a CTL response, while on the other hand, use of a minimal CTL antigen (such as a nonamer peptide) which does not efficiently drive an antibody response, has also failed to induce a CTL response. Attempts to develop adjuvants that enhance the immunostimulatory aspects of minimal CTL antigens have resulted in the production of materials (i.e. adjuvants) that also induce a competing humoral immune response, or, which simply offer little CTL stimulatory effect.

It has also been suggested that certain controlled release technology using microspheres or liposomes with subunit antigens and peptides might be effective to enhance immunogenecity. The combination of sustained release and depot effect is suggested to reduce the amount of antigen needed and eliminate booster shots. However, the preparation of such compositions is difficult and unpredictable, and vaccine formulations based on this technology have not been translated into effective clinical treatments.

As can be seen from the foregoing, there has been little success at developing a CTL vaccine that is both capable of inducing a strong CTL response then sustaining that response over time. The development of a vaccine with these capabilities is essential before effective anti-tumor therapy based on CTL immunity can be contemplated.

OBJECTS OF THE INVENTION

An object of this invention is to provide a method for inducing or sustaining a specific CTL immunological response in a mammal over time.

Another object of this invention is to provide a method for treating a mammal having a malignant tumor or infectious disease by inducing and sustaining an immunological attack on the malignant tumor or infectious disease in the mammal.

It is a further object of this invention to provide an article of manufacture useful for inducing and sustaining a specific immunological CTL response in a mammal over time.

It is a further object of this invention to provide an article of manufacture useful for treating a mammal having a malignant tumor or infectious disease, which article is designed to induce and maintain an immunological attack on the malignant tumor or infectious disease in the mammal.

It is a further object of this invention to provide a portable device for sustained delivery of an antigen to a mammal having a malignant tumor or infectious disease, where the antigen stimulates the mammal's immune system to attack the tumor or infectious disease and the device is located outside the mammal.

It is still a further object of this invention to provide an implantable device for sustained delivery of an antigen to a mammal having a malignant tumor or infectious disease, where the antigen stimulates the mammal's immune system to attack the tumor or infectious disease.

It is a further object of this invention to provide antigen compositions and containers therefor that are useful in the methods, devices, and/or articles of manufacture of this invention.

Other objects of this invention may be apparent to those of skill in the art by reading the following specification and claims.

SUMMARY OF THE INVENTION

In one aspect of the invention, a method is provided for inducing an immunological CTL response to an antigen by sustained, regular delivery of the antigen to a mammal so that the antigen reaches the lymphatic system. In particular, the antigen is delivered to the mammal at a level sufficient to induce an immunologic CTL response in the mammal and the level of the antigen in the mammal's lymphatic system is maintained over time sufficient to maintain the immunologic CTL response. Preferably, the antigen is delivered directly to the mammal's lymphatic system, such as to the spleen, a lymph node or lymph vessel.

Also provided is a method of treating an animal having a disease, or being predisposed to a disease, to which the animal's immune system mounts a cell-mediated response to a disease-related antigen to attack the disease. In this aspect of the invention, a disease-matched antigen is delivered to the animal at a level sufficient to induce an increased CTL-response in the animal which is then maintained in the animal by sustained, regular delivery of the disease-matched antigen to the animal for a time sufficient to treat the disease. The sustained, regular delivery of the antigen is done in a manner that maintains the level of antigen in the animal's lymphatic system. Preferably, the sustained, regular delivery is achieved by pumping a physiologically-acceptable, composition of the antigen from a device held external of or implanted in the animal's body so that the antigen reaches the animal's lymph system. Optionally, a cytokine that is capable of enhancing the CTL response is delivered and/or maintained along with the antigen. Diseases addressed in this manner include cancer and pathogenic diseases.

In a further aspect of the invention, an article of manufacture is provided for delivering an antigen that induces a CTL response in an animal. In particular, the article comprises a reservoir of a physiologically-acceptable, antigen-containing composition that is capable of inducing a CTL response in an animal; a pump connected to the reservoir to deliver the composition at a defined rate; a transmission line to discharge the composition from the reservoir; and, optionally, a delivery line connected to the transmission line, which delivery line is of a size suitable for positioning in the animal and for delivery of the composition in a manner that reaches the lymphatic system of the animal.

In a further aspect of the invention, a process is provided for preparing a system useful for inducing a sustained CTL response in an animal needing such a response, which comprises placing a physiologically-acceptable, antigen-containing composition in a reservoir having a pump for delivering the composition at a defined rate through a transmission line to the animal.

Another aspect of the invention is a method of inducing and/or sustaining an immunological CTL response in a mammal by delivering an antigen in the form of a polypeptide directly to the lymphatic system of the mammal. The antigen can be delivered at a level sufficient to induce an immunologic CTL response in the mammal and the level of the antigen in the mammal's lymphatic system is preferably maintained over time sufficient to maintain the immunologic CTL response.

The antigen can be an 8-10 amino acid peptide. Further, the peptide sequence can be derived from a tumor-associated antigen. Examples of tumor-associated antigens include MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGE), SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, and the like.

The peptide sequence also can be derived from a microbial antigen. Further, the antigen can be provided as a component of a microorganism or mammalian cell. Examples of microorganisms include a protozoan, a bacterium, a virus, and the like; the mammalian cell can be an antigen presenting cell, such as, for example, a dendritic cell.

The antigen can be a native component of the microorganism or mammalian cell. The microorganism or mammalian cell can include, for example, an exogenous antigen. Also, the microorganism or mammalian cell can include a recombinant nucleic acid encoding or promoting expression of the antigen. The microorganism or mammalian cell can express a tumor-associated antigen, or a microbial antigen native to a second microbial species. The antigen can be provided as an 8-10 amino acid peptide.

The present invention in another aspect includes a method of inducing and/or sustaining an immunological CTL response in a mammal by delivering an antigen, in the form of a vector that can include a nucleic acid encoding the antigen, directly to the lymphatic system of the mammal. The antigen can be delivered at a level sufficient to induce an immunologic CTL response in the mammal and the level of the antigen in the mammal's lymphatic system is preferably maintained over time sufficient to maintain the immunologic CTL response.

The vector can be a plasmid and the like. The vector further can include a bacterium and the like. The bacterium, for example, can include Listeria, Shigella, Salmonella, Escherichia, and the like. The vector, for example, can be a virus, such as, for example, pox viruses, adenoviruses, adeno-associated viruses, retroviruses, herpesviruses, and the like.

The nucleic acid can encode, for example, a tumor-associated antigen. Examples of tumor-associated antigens include MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGE), SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, and the like.

The nucleic acid can encode, for example, a microbial antigen. Examples of microbial antigens include a viral antigen, a bacterial antigen, a protozoal antigen, and the like. The nucleic acid can encode, for example, a protein or other polypeptide. The nucleic acid also can encode an 8-10 amino acid peptide.

The nucleic acid can be plasmid DNA in a formulation comprising about 1-10% ethyl alcohol, 0-1% benzyl alcohol, 0.25-0.5 mM EDTA and a citrate-phosphate buffer of pH 7.4-7.8, comprising about 3-50 mM citrate and about 90-200 mM phosphate. For example, the formulation can include 1% ethyl alcohol, 1% benzyl alcohol, 0.5 mM EDTA and a citrate-phosphate buffer of pH 7.4 to 7.8 comprising 50 mM citrate and 100 mM phosphate.

The invention in another aspect provides a method of inducing and/or sustaining an immunological CTL response in a mammal by delivering a microorganism or mammalian cell directly to the lymphatic system of the mammal. The microorganism or mammalian cell are preferably delivered at a level sufficient to induce an immunologic CTL response in the mammal and the level of the microorganism or mammalian cell in the mammal's lymphatic system is preferably maintained over time sufficient to maintain the immunologic CTL response.

A further aspect of the invention is a method of inducing and/or sustaining an immunological CTL response in a mammal by delivering a nucleic acid, capable of conferring antigen expression, directly to the lymphatic system of the mammal. The nucleic acid can be delivered at a level sufficient to induce an immunologic CTL response in the mammal and the level of the nucleic acid in the mammal's lymphatic system is preferably maintained over time sufficient to maintain the immunologic CTL response.

A further aspect of the invention is a method of inducing and/or sustaining an immunological CTL response in a mammal by delivering a non-peptide antigen directly to the lymphatic system of the mammal. The antigen is preferably delivered at a level sufficient to induce an immunologic CTL response in the mammal and the level of the antigen in the mammal's lymphatic system is preferably maintained over time sufficient to maintain the immunologic CTL response.

The invention also provides an article of manufacture for delivering an antigen that induces a CTL response in an animal. In particular, the article can be an external device. The article can include a reservoir of a physiologically-acceptable, antigen-containing composition that can be capable of inducing a CTL response in an animal, a pump connected to the reservoir to deliver the composition at a defined rate, a transmission line to discharge the composition from the reservoir; and, a delivery line connected to the transmission line. The delivery line can include a catheter of at least 20 mm for positioning in the animal and for delivery of the composition to the lymphatic system of the animal.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings in which:

FIG. 1 is a graph showing the lysis of target cells by CTL versus the effector/target ratio when antigen is delivered as a single dose (circles) and when antigen is delivered by a continuous pump (triangles).

FIG. 2 (A and B) are graphs showing the lysis of target cells by CTL versus the effector/target ratio when antigen is delivered as a single dose (circles), when antigen is delivered by a continuous pump (triangles) and negative control (squares) at (A) 36 hours and (B) 7 days.

FIG. 2C is a graph showing the footpad swelling versus time when antigen is delivered as a single dose (circles) and when antigen is delivered by a continuous pump (triangles).

FIG. 3 is a graph showing the lysis of target cells by CTL versus the dose of the peptide antigen when the antigen is delivered subcutaneously, intravenously and intrasplenically.

FIG. 4 is a bar graph showing tritiated thymidine uptake in CTL cells induced by antigen introduced intravenously, intrasplenically and subcutaneously.

FIG. 5 is a rough schematic of a human lymphatic system.

FIG. 6. Comparison of anti-peptide CTL responses following immunization with various doses of DNA by different routes of injection.

FIG. 7. Comparison of anamnestic antiviral CTL responses following immunization with various doses of DNA by different routes of injection.

FIG. 8. Protective immunity against systemic and peripheral virus infection following intra-lymph node immunization with DNA. LCMV titer in spleen (A) and Vacc-G2 vaccinia titers in ovary (B) following indicated immunization and subsequent viral challenge.

FIG. 9. Growth of transplanted gp33 expressing tumor in mice immunized by i.ln. injection of gp33 epitope-expressing, or control, plasmid.

FIG. 10. Amount of plasmid DNA detected by real-time PCR in injected or draining lymph nodes at various times after i.ln. of i.m. injection, respectively.

FIG. 11. Average % supercoiled DNA in formulations 1-9 over 7 days.

DETAILED DESCRIPTION OF THE INVENTION

Method of Treatment

One aspect of this invention is a method for inducing or sustaining a specific immunological response (i.e., a CTL response) in an animal that has a disease (or predisposition to a disease) in which the animal's immune system may attack the disease with a natural CTL response. The response and diseases are discussed in greater detail hereinafter. The method has particular value for treating an animal having a malignant tumor in order to inhibit the growth of the tumor or for treating a chronic infectious disease such as hepatitis or AIDS.

The method, along with other aspects of the invention, is useful in an animal having an immune system that includes a lymphatic system. This generally includes vertebrates, specifically mammals and particularly humans. Thus, this invention will find use in treating humans of all ages as well as in treating animals, i.e. in veterinary uses. The invention may be used for treating livestock such as cattle, sheep, pigs, goats, and the like or for treating household pets such as dogs, cats, rabbits, hamsters, mice, rats, and the like. The primary use will be for treating humans that are in need of having a specific immunological response sustained for treatment of a disease such as cancer or chronic infections.

A key aspect of this invention is the delivery of an appropriate antigen to the lymphatic system of the animal being treated and sustaining the delivery over time. This is based in part on the observation that a strong induction and a sustained CTL response require ongoing antigenic stimulation of the lymphatic system. In a human, the lymphatic system includes lymph, lymphocytes, lymph vessels, lymph nodes, tonsils, the spleen, the thymus gland, and bone marrow. The lymphatic system performs three basic functions. First, it helps maintain fluid balance in the tissues. Approximately 30 L of fluid pass from the blood capillaries into the interstitial spaces each day, whereas only 27 L pass from the interstitial spaces back into the blood capillaries. If the extra 3 L of interstitial fluid were to remain in the interstitial spaces, edema would result, causing tissue damage and eventual death. These 3 L of fluid (i.e. lymph) enter the lymph capillaries, then passes through the lymph vessels to return to the blood. Lymph is similar in composition to plasma. In addition to water, lymph contains solutes derived from two sources: (1) substances in plasma such as ions, nutrients, gases, and some proteins pass from blood capillaries into the interstitial spaces to become part of the lymph; and (2) substances derived from cells within the tissues such as hormones, enzymes, and waste products are also found in lymph.

The lymphatic system's second basic function is to absorb fats and other substances from the digestive tract. Special lymph vessels called lacteals are in the lining of the small intestine. Fats enter into the lacteals and pass through the lymph vessels to the venous circulation. The lymph passing through these capillaries has a milky appearance because of its fat content, and it is called chyle.

The third basic function of the lymphatic system is to act as part of the body's defense system. The lymph nodes filter lymph, and the spleen filters blood, removing microorganisms and other foreign substances. This third function is the function most important to this invention in that the antigen must be delivered to the lymph system at a level sufficient to elicit the desired, specific immunological response in the animal. FIG. 5 is a schematic representation of a human lymphatic system showing the major lymphatic organs and vessels.

As hereinbefore mentioned, the present invention relates to a method of inducing or sustaining a specific immunological response (particularly a CTL response) to an antigen in an animal over time. The method comprises delivering the antigen to the animal in a manner that delivers the antigen into the lymphatic system of an animal to sustain the desired response over time. Generally this is done by establishing a mechanism to transfer an antigen from a reservoir to the animal's lymphatic system on a regular basis over time. The antigen may be delivered by a variety of methods that target intralymphatic presentation, including subcutaneous injection, direct injection into the lymphatic system by an antigen delivery vehicle that is implanted, preferably at or near a lymphatic organ, or by an antigen delivery vehicle that is external to the animal but contains a means (e.g. a needle or catheter) to deliver the antigen into the lymphatic system. By this method one can avoid multiple ongoing injections and can also avoid the use of including professional antigen-presenting cells in the composition held in the reservoir.

The method of this invention can be viewed as inducing CTL immune response by providing high continuous local concentrations of antigen, which otherwise is quickly removed and degraded from the body after bolus injection. Potent activation of CD8+ T cells requires signaling through the T cell receptor (TCR) in a manner that is dependent on both quantitative and qualitative factors. Quantitative factors refer to the number of TCRs engaged by peptide-MHC complexes. Qualitative considerations include the duration of engagement of the TCR by peptide-MHC complexes, with specific peptide-MFIC complexes. Sustained regular deliveries of antigen allows optimal conditions to be established for inducing CD8+ T cells.

The antigen is delivered to the animal so that the antigen is present in the animal's lymphatic system on a sustained basis over a period of time. That is to say, it is delivered in such a way that the presence of the antigen is maintained over the period of time in the animal's lymphatic system. Thus, the antigen is delivered to the animal on a regular basis, i.e. the antigen is delivered regularly without significant interruption over the period of time. This regular delivery is achieved by the constant delivery of the antigen at low levels directly to the lymphatic system using an external device or an implantable device, as discussed hereinafter. Alternatively, the antigen can be delivered at higher levels to the animal by subcutaneous injection with indirect absorption or equilibration with the lymph system. Delivery on a regular basis is meant to include intermittent (stopping and transmitting at intervals) as well as continuous (transmitting without interruption) delivery. In intermittent delivery, the times transmission is stopped will not be enough to reduce the level of antigen in the animal's lymphatic system to eliminate the desired specific immunological response. Thus, the antigen may be delivered in pulses or small doses over time.

Preferably, the sustained delivery is achieved by the positioning of a means of delivery so that the animal being treated does not have to receive multiple injections of the antigen, but instead has only one insertion of the means for delivery, e.g. an insertion of a catheter or needle for infusion of a suitable antigen-containing composition or the surgical implantation of an implantable device that releases an appropriate, antigen-containing composition on a sustained basis.

The period of time over which the antigen will be released will be a time sufficient to induce and maintain the desired specific immunological response, e.g. to maintain a CTL response, and in the case of an animal with a tumor or infection, at a level sufficient to stimulate the immune system to attack the tumor and inhibit its growth or to attack the infection. Generally, this period of time may vary from a few days, e.g. a week, to a year or more. Preferably, the treatment, i.e. sustained delivery of the antigen, will extend for at least seven days and no more than six months. It has been found that the CTL response is induced by administration for at least seven days. To determine the period of time, the attending physician will evaluate, i.e., the severity of the condition, the strength of the patient, the antigenic response (e.g., the level of CD8+ cells measurable in the patient's system), the presence of toxic effects, and other factors known to one of skill in the art. Ultimately the time for sustained delivery in a cancer patient will be that necessary for improvement in the patient as evidenced by reduction in the size of the tumor, the rate of growth of the tumor, and/or the improvement in the overall health of the patient being treated. In the treatment of infectious diseases the treatment is continued until the health of the patient improves sufficiently to stop treatment.

The underlying immunological rationale for the utility of this invention arises from certain immunological considerations, The immune system has evolved to protect the host from microbial infection. CD4+ T cells together with B cells are the main components of the immune system humoral effector arm, which is crucial to eliminate extracellular pathogens or toxins. In contrast, the CD8+ T cell arm of the immune system is mainly responsible for eliminating intracellular pathogens, i.e. most importantly viruses, either via cytokine release or by cytotoxic activity. It is now emerging that these most efficient “killer cells” of the immune system would best serve as the primary effector cells in tumor immunotherapy. An object of this invention is to mount a disease-specific CTL response (CD8+ T cell response) against the disease and sustain it over time, e.g., a tumor specific or microbial specific CTL response.

CD8+ T cells recognize antigenic oligopeptides presented on HLA class I molecules of target cells, e.g., tumor cells. The sequences of many HLA-A1 and HLA-A2 presented tumor and pathogen specific antigen peptides have recently been characterized. These peptides may be used in this invention to induce, e.g., a melanoma-specific CD8+ T cell response. These peptides are discussed hereinafter.

In contrast to viral infection, class I-binding oligopeptides show only low immunogenicity. Most viruses induce peak CD8+ T cell responses around 7-10 days after systemic spread. This invention aims at enhancing the immunogenecity of class I binding oligopeptides by sustained, regular release of peptide into a lymphatic system and continued release into the lymphatic system.

In contrast to antibody-mediated B cell memory, which is long lived, T cell memory appears to be short lived or non-existent. In accordance with this invention, maintenance of functional T cell memory depends on persistence of antigen through continued, regular administration of the desired antigen. Having made this invention and looking at past concepts that might support this underlying rationale, some evidence includes the observation that delayed type hypersensitivity (DTH) of the tuberculin type (the only functional test for T cell memory in humans), can be elicited only in granulomatous disease, such as tuberculosis (tuberculin test), leprosy (lepromin test), brucellosis (brucellin test), sarcoidosis (Kveim test), Histoplasmosis (histoplasimin test) etc., but no such test could be established for non-granulomatous infectious disease. A factor that all granulomatous diseases have in common, is that the antigen persists within the granuloma—professional antigen presenting cells can use this reservoir to continuously restimulate specific T cells in lymphoid organs. In mice models (see Example 3) it is demonstrated that maintenance of functional CD8+ T cell memory was strictly dependent on continuous antigenic restimulation.

To determine whether a CTL response is obtained in an animal being treated in accordance with this invention, one measures the level of CD8+ cells (i.e. CTL) present in the blood or lymphatic organs such as the spleen or lymph nodes. This determination is done by first measuring the level of CD8+ cells before performing the method of this invention and measuring the level during treatment, e.g. at 7, 10, 20, 40 days, etc. The level or strength of the CD8+ (CTL) response can be assessed in vivo or in vitro. In humans, there exists so far only one in vivo test to measure CD8+ T cell responses, which is a skin test. In this skin test, HLA class I binding peptides are injected, intradermally (such as described in Jäger, E. et al. Granulocyte-macrophage-colony-stimulating Factor Enhances Immune Responses To Melanoma-associated Peptides in vivo Int. J Cancer 67, 54-62 (1996)). If a CTL response is present, these cells will recognize and attack peptide pulsed dermal cells, causing a local inflammatory reaction either via cytokine release or the cytotoxic mechanism (Kündig, T. M., Althage, A., Hengartner, H. & Zinkernagel, R. M. A skin test to assess CD8+ cytotoxic T cell activity. Proc. Natl. Acad Sci. USA 89:7757-776 (1992)). This inflammatory reaction can be quantified by measuring the diameter of the local skin rash and/or by measuring the diameter of the infiltrate (i.e., the swelling reaction). As an alternative to the injection of soluble free peptide, the HLA-class I binding peptide can also be injected intradermally in a bound form, e.g., bound to extracorporally derived dendritic cells. In other mammals, additional, although experimental, in vivo tests to assess CD8+ T cell responses exist. For example, in a mouse model, CD8+ T cell responses can be measured by challenge infection with a vaccinia recombinant virus expressing the peptide used for immunization. While naïve mice succumb to the infection with the vaccina recombinant virus, mice with preexisting CD8+ T cell immunity against the peptide epitope expressed by the vaccinia recombinant virus, are immune to reinfection. The level of immunity to reinfection can be quantified as the factor of reduction of the vaccinia virus titer recovered from mouse organs after challenge infection (Bachmann, M. F. & Kundig, T. M. In vitro vs. in vivo assays for the assessment of T- and B-cell function. Curr. Opin. Immunol. 6, 320-326 (1994)). For example, 5 days after challenge infection, a typical vaccinia recombinant virus titer recovered from a mouse ovary would be around 10⁷ pfu per ovary, whereas the vaccinia recombinant virus titer in a mouse with a preexisting CD8+ T cell response against the recombinant gene product would for example be around 10³ pfu per ovary. Such a 10,000 fold-reduction in virus titer reflects biologically significant preexisting CD8+ T cell activity against the recombinant gene product.

The level of CD8+ T cell responses can also be quantified in vitro, by estimating the number of CD8+ T cells specific for the antigenic peptide in question. In a naïve mammal the so called “frequency”, i.e., the number of specific CD8+ T cells divided by the number of non-specific white blood cells, is less than 10⁻⁶. After successful immunization, the frequency increases due to proliferation of specific T cells. During an acute viral infection, for example, the frequency of specific CD8+ T cells may rise to 10⁻². Then, after elimination of the virus, the frequency of specific CD8+ T cells usually drops to a “memory” level of around 10⁻⁴. Thus, the CD8+ T cell response can be quantified by measuring the frequency of specific CD8+ T cells. The higher the frequency, the stronger the response. The classical assays used to measure the frequency of specific CD8+ T cells are based on limiting dilution cell culture techniques, as described in detail by Kündig, T. M. et al. (On the role of antigen in maintaining cytotoxic T cell memory. Proceedings of the National Academy of Sciences of the United States of America 93, 9716-972′) (1996)). A novel approach to estimate the frequency of specific CD8+ T cells is to construct soluble class I MHC (for use in mice) or HLA molecules (for use in humans) with a peptide bound to their groove, so that the specific T cell receptors will bind to these complexes. These complexes can be labeled for detection, for example, with a fluorescent substance, allowing for detection by flow cytometry.

One current procedure to render peptides immunogenic is to inject them in context with “nature's most potent adjuvant”, i.e., professional antigen presenting cells (APCs) such as dendritic cells (DCs), (Steinmann, R. M., The dendritic cells system and its role in immunogenicity, Annual Review of Immunology 9, 271-96 (1991)). DCs are the most potent APCs of the immune system. They can now be cultured in vitro by adding granulocyte macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha) or interleukin-4 (IL-4) to progenitors isolated from the blood of patients or mice (Inaba, K. et al., Identification of proliferating dendritic cell precursors in mouse blood, Journal of Experimental Medicine 175, 1157-1167 (1992)). Large numbers of DCs can then be pulsed with tumor specific antigen peptides and are injected back into the patient, where they migrate into lymphatic organs to induce T cell responses (Young, J. W. & Inaba, K., Dendritic Cells As Adjuvants For Class I Major Histocompatibility Complex-restricted Anti-tumor Immunity, Journal of Experimental Medicine 183, 7-11 (1996)). An object of this invention is to circumvent the time-consuming, labor intensive procedure of culturing DCs after isolation of DC progenitors and deliver the antigen to the lymphatic system free of APCs such as DCs. The method of this invention, i.e., the sustained, regular delivery of antigen into a lymphatic organ, allows sufficiently high local concentrations of antigen inside the lymphatic organ, such that professional antigen presenting cells, e.g., dendritic cells, can be loaded with peptide in vivo. This can be viewed as a method of loading antigen presenting cells (dendritic cells) in vivo for inducing a CTL response.

The method of the present invention is clearly advantageous over the prior art methods for inducing a CTL response against a tumor or virus. For example, the present invention does not require repetitive immunizations to effect for prolonged anti-tumor immunotherapy. The sustained delivery of the antigen maintains the CTL response that could ultimately afford a prolonged aggressive posture of CTL against tumor cells, more thorough eradication, and protection against recurrence during the vaccine treatment. In the absence of antigen, CTL that have undergone primary activation soon cease to recirculate through the body, soon finding their way to the spleen where they become quiescent. Since CTL must immediately deliver a lethal hit, their residence in the spleen precludes an active role in protection against infections or tumor growth at distant sites in the body. The controlled release of antigen recognized by CTL in this invention circumvents this outcome as antigen delivery is maintained. Sustained released antigen delivery to the lymphatic system by this invention solves two major problems: it provides for potent CTL stimulation that takes place in the milieu of the lymphoid organ, and it sustains stimulation that is necessary to keep CTL active, cytotoxic and recirculating through the body.

Another fundamental improvement of the present method over prior art is that it facilitates the use of inherently non-immunogenic peptide antigens for CTL stimulation without the combined use of conventional adjuvants. This is very beneficial as most experimental adjuvants are toxic and poorly suited for use in humans. In addition adjuvants stimulate the TH2-type humoral immune response that negatively affects the CTL response. Further, since conventional adjuvants are not required, only the minimal antigenic epitope for a CTL response is required in the formulation.

An additional advantage to the method of the present invention, where it embodies the use of mechanical delivery systems, is that the antigen delivery can be stopped if any adverse immunological effects are observed, For example, in vaccines against melanoma, CTL have been induced to attack not only malignant melanocytes but also healthy tissue, causing “vitiligo.” The ability to discontinue a CTL vaccine at any time is a significant advance in vaccine safety. Peptides have a short half-life due to catabolism in the liver. Therefore, the stimulation-effect falls soon after cessation of delivery.

As pointed out before, the method of this invention has two parts: (1) inducing an increased CTL response and (2) maintaining the response. The inducing and maintaining may be performed using the same device, as discussed hereinafter, or the inducing may be done separately, e.g., by a separate injection of an antigen then following up with sustained delivery of the antigen over time to maintain the response.

Diseases Treated According to the Invention

In general, this invention is useful for treating an animal having (or being predisposed to) any disease to which the animal's immune system mounts a cell-mediated response to a disease-related antigen in order to attack the disease. Thus, the type of disease may be a malignant tumor or a chronic infectious disease caused by a bacterium, virus, protozoan, helminth, or other microbial pathogen that enters intracellularly and is attacked, i.e., by the cytotoxic T lymphocytes, In addition, the invention is useful for treating an animal that may be at risk of developing such diseases.

Malignant Tumors

In a mature animal, a balance usually is maintained between cell renewal and cell death in most organs and tissues. The various types of mature cells in the body have a given life span; as these cells die, new cells are generated by the proliferation and differentiation of various types of stem cells. Under normal circumstances, the production of new cells is so regulated that the numbers of any particular type of cell remain constant. Occasionally, though, cells arise that are no longer responsive to normal growth-control mechanisms. These cells give rise to clones of cells that can expand to a considerable size, producing a tumor, or neoplasm. A tumor that is not capable of indefinite growth and does not invade the healthy surrounding tissue extensively is benign. A tumor that continues to grow and becomes progressively invasive is malignant; the term cancer refers specifically to a malignant tumor. In addition to uncontrolled growth, malignant tumors exhibit metastasis; in this process, small clusters of cancerous cells dislodge from a tumor, invade the blood or lymphatic vessels, and are carried to other tissues, where they continue to proliferate. In this way a primary tumor at one site can give rise to a secondary tumor at another site. The methods, devices and articles of manufacture discussed herein are useful for treating animals having malignant tumors.

Malignant tumors treated according to this invention are classified according to the embryonic origin of the tissue from which the tumor is derived. Carcinomas are tumors arising from endodermal or ectodermal tissues such as skin or the epithelial lining of internal organs and glands. A melanoma is a type of carcinoma of the skin for which this invention is particularly useful. Sarcomas, which arise less frequently, are derived from mesodermal connective tissues such as bone, fat, and cartilage. The leukemias and lymphomas are malignant tumors of hematopoietic cells of the bone marrow. Leukemias proliferate as single cells, whereas lymphomas tend to grow as tumor masses. The malignant tumors may show up at numerous organs or tissues of the body to establish a cancer. The types of cancer that can be treated in accordance with this invention include the following: bladder, brain, breast, cervical, colo-rectal, esophageal, kidney, liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach, uterine, and the like. The present invention is not limited to the treatment of—an existing tumor or infectious disease but can also be used to prevent or lower the risk of developing such diseases in an individual, ie., for prophylactic use. Potential candidates for prophylactic vaccination include individuals with a high risk of developing cancer, i.e., with a personal or tuminal history of certain types of cancer.

The incidence of skin cancer has increased substantially over the last decades. Lifetime analysis indicates that around 1/1500 humans born in 1935, 1/600 born in 1960, 1/100 born in 1990 and a projected 1/75 humans born in the year 2000 will have melanoma in their lifetime. Surgical excision usually cures melanoma. However, even small looking lesions may have already metastasized at the time of diagnosis. The prognosis of metastasized melanoma is very poor and correlates with the thickness of the primary tumor and with its localization.

The current treatment of malignant melanoma aims at surgical removal of the primary tumor. If metastases are present, chemotherapy and biological response modifiers are additionally used. However, patients with stage IV malignant melanoma are almost invariably incurable and treatments are palliative. Patients with Stage 1V malignant melanoma have a median survival time of approximately one year and only a 10% chance of long-term survival. There is at present no generally accepted standard therapy for metastatic melanoma. Objective response rates to mono- or polychemotherapy are low in comparison with other tumors, reaching no more than 15-35%. An improved treatment outcome in stage IV malignant melanoma seems unachievable either by chemotherapeutic combinations or by increasing doses to levels where autologous bone marrow transplantation becomes necessary. The method of this invention is useful for treating malignant melanoma, even at Stage IV.

Infectious Diseases

Infectious diseases, which have plagued animal populations (particularly humans) throughout-history, still cause millions of deaths each year. The infectious diseases that can be treated using this invention include those caused by pathogens such as bacteria, viruses, protozoa, helminths, and the like. These diseases include such chronic diseases such as acute respiratory infections, diarrheal diseases, tuberculosis, malaria, hepatitis (hepatitis A, B C, D, E, F virus), measles, mononucleosis (Epstein-Barr virus), whooping cough (pertussis), AIDS (human immunodeficiency virus I & 2), rabies, yellow fever, and the like. Other diseases caused by human papilloma virus or various strains of virus are treatable by this method.

In some instances, the mammal, in particular human, can be treated prophylactically, such as when there may be a risk of developing disease. An individual travelling to or living in an area of endemic infectious disease may be considered to be at risk and a candidate for prophylactic vaccination against the particular infectious agent. For example, the CTL response can be induced in a human expecting to enter a malarial area and/or while in the malarial area by using a CTL-inducing, -malaria-specific antigen to lower the risk of developing malaria. Preventative treatment can be applied to any number of diseases including those listed above, where there is a known relationship between the particular disease and a particular risk factor, such as geographical location or work environment.

Antigens Useful in the Invention

An antigen useful in this invention is one that stimulates the immune system of a mammal having a malignant tumor or infectious disease to attack the tumor and inhibit its growth or to destroy the pathogen causing the disease. Thus, the antigen used in the invention is matched to the specific disease found in the animal being treated. In this regard the antigen may be said to induce a CTL response (also referred to as a cell-mediated immune response), i.e. a cytotoxic reaction by the immune system that results in lysis of the target cells (e.g., the malignant tumor cells or pathogen-infected cells).

To determine whether an antigen is matched to a particular patient, whether human or other animal, the tissue type of the patient is first determined. If human, the tissue must demonstrate the appropriate human leukocyte antigen (HLA) capable of binding and displaying the antigen to CTL. It is preferable that the HLA typing be performed, on the target cells, since a significant portion of tumors escape immune detection by downregulating expression of HLA. Therefore HLA expression on normal cells of the patient does not necessarily reflect that found on tumor cells in their body. A tumor from a patient is also screened to determine if he or she expresses the antigen that is being used in the vaccine formulation. Immunohistochemistry and/or polymerase chain reaction (PCR) techniques both can be used to detect antigen in the tumor cells. Immunohistochemistry offers the advantage in that it stains a cross-section of tumor in a slide preparation, allowing investigators to observe the antigen expression pattern in cross-section of tumor, which is typically heterogeneous for antigen expression. PCR has the advantage of not requiring specific monoclonal antibodies for staining and is a fast and powerful technique. In addition, PCR can be applied in situ. Ideally, both immunohistochemical and PCR methods should be combined when assessing antigen expression in tumors. While the antigen compositions useful in this invention are designed to include the most commonly expressed tumor antigens (as discussed hereafter), not all tumors will express the desired antigen(s). Where a tumor fails to express the desired antigen, the patient is excluded for consideration for that particular antigen composition. Thus, an aspect of this invention is a process for preparing a device useful for providing a sustained CTL response over time by matching a subject's antigen specific to the tumor or pathogen in the subject, preparing a physiologically-acceptable composition of the antigen so matched, and combining the composition in a suitable delivery device as discussed in hereinafter.

Immune activation of CD8+ T cells generates a population of effector cells with lytic capability called cytotoxic T lymphocytes, or CTL. These effector cells have important roles in the recognition and elimination of malignant cells and pathogens. In general, CTL are CD8+ and are therefore class I MHC restricted, although in rare instances CD4+ class II-restricted T cells have been shown to function as CTL. Since virtually all nucleated cells in the body express class I MHC molecules, CTL can recognize and eliminate almost any altered body cell. CD8+ T cells recognize antigen presented on HLA class I molecules of tumor cells through T cell receptors.

The CTL-mediated immune response can be divided into two phases, reflecting different aspects of the cytotoxic T-cell response. The first phase involves the activation and differentiation of T_(c) (CD8+) cells into functional effector CTLs. In the second phase, CTLs, recognize antigen—class I MHC complexes on specific target cells, initiating a sequence of events that culminates in target-cell destruction. Further detailed discussion of the process is found at Chapter 15 of the Second Edition of “Immunology” by Janis Kuby, W.H. Freeman and Company (1991).

The type of tumor antigen used in this invention may be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA), A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA associated antigen is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells. TSAs and TAAs can be jointly referred to as TRA or a tumor related antigen.

Tumor antigens useful in the present invention, whether tumor-specific or tumor-associated, must be capable of inducing a CTL-mediated immune response. The presence of tumor antigens that elicit a cell-mediated response has been demonstrated by the rejection of tumors transplanted into syngeneic recipients; because of this phenomenon, these tumor antigens are referred to as tumor-specific transplantation antigens (TSTAs) or tumor-associated transplantation antigens (TATAs). It has been difficult to characterize tumor transplantation antigens because they do not generally elicit an antibody response and therefore they cannot be isolated by immunoprecipitation. Many are peptides that are presented together with MHC molecules on the surface of tumor cells and have been characterized by their ability to induce an antigen-specific CTL.

The type of pathogen specific antigen useful in this invention may be short oligopeptides derived from pathogen proteins. These oligopeptides must bind to class I MHC (for use in mice), class I HLA (for use in humans), or class I molecules of any other mammals. Also, such class I molecule bound peptides should be recognizable by specific T cell receptors. Such oligopeptides usually have a length of 8-15 amino acids. Several examples of such pathogen derived oligopeptides, so called T cell epitopes, are given in Tables I and II.

The tumor antigens and pathogen-specific antigens useful in this invention are generally thought to be presented at the surface of an antigen presenting cell (APC) to stimulate the immune system through class I molecules of the major histocompatability complex (MHC) interactively with the CD8+ cells.

Antigens useful in the invention are generally protein-based entities of a molecular weight of up to 100,000 daltons. Appropriate antigens include, but are not limited to differentiation antigens, tumor-specific multilineage antigens, embryonic antigens, antigens of oncogenes and mutated tumor-suppressor genes, unique tumor antigens resulting from chromosomal translocations, viral antigens, and others that may be apparent presently or in the future to one of skill in the art. It is preferable that the antigen be a peptide of 8 to 15 amino acids in length that is an epitope of a larger antigen, i.e. it is a peptide having an amino acid sequence corresponding to the site on the larger molecule that is recognized and bound by a particular T-cell receptor. These smaller peptides are available to one of skill in the art by following the teachings of U.S. Pat. Nos. 5,747,269 to Rarnmensee et al. issued May 5, 1998; 5,698,396 to Pfreundschuh issued Dec. 16, 1997; and PCT Application Numbers PCT/EP95/02593 filed 4 Jul. 1995, PCT/DE96/00351 filed 26 Feb. 1996, all of which are incorporated herein by reference. Additional approaches to epitope discovery are described in U.S. Pat. No. 6,037,135 METHODS FOR MAKING HLA BINDING PEPTIDES AND THEIR USES and U.S. patent application Ser. No. 09/561,074 entitled METHOD OF EPITOPE DISCOVERY both of which are incorporated herein by reference in their entirety.

While in the general case the antigen ultimately recognized by a T cell is a peptide, it must be kept in mind that the form of antigen actually administered as the immunogenic preparation need not be a peptide per se. When administered, the epitopic peptide(s) may reside within a longer polypeptide, whether the complete protein antigen, some segment of it, or some engineered sequence. Included in such engineered sequences would be polyepitopes and epitopes incorporated into some carrier sequence such as an antibody or viral capsid protein. Such longer polypeptides may include epitope clusters as described in U.S. patent application Ser. No. 09/561,571 entitled “EPITOPE CLUSTERS,” which is incorporated herein by reference in its entirety. The epitopic peptide, or the longer polypeptide in which it is contained, may be a component of a microorganism (e.g. a virus, bacterium, protozoan, etc.), or a mammalian cell (e.g. a tumor cell or antigen presenting cell), or lysates, whole or partially purified, of any of the foregoing. They may be used as complexes with other proteins, for example heat shock proteins. The epitopic peptide may also be covalently modified, such as by lipidation, or made a component of a synthetic compound, such as dendrimers, multiple antigen peptides systems (MAPS), and polyoximes, or may be incorporated into liposomes or microshperes, etc. As used in this disclosure the term “polypeptide antigen” encompasses all such possibilities and combinations. The invention comprehends that the antigen may be a native component of the microorganism or mammalian cell. The antigen may also be expressed by the microorganism or mammalian cell through recombinant DNA technology or, especially in the case of antigen presenting cells, by pulsing the cell with polypeptide antigen prior to administration. Additionally, the antigen may be administered encoded by a nucleic acid that is subsequently expressed by APCs. Finally, whereas the classical class I MHC molecules present peptide antigens, there are additional class I molecules which are adapted to present non-peptide macromolecules, particularly components of microbial cell walls, including without limitation lipids and glycolipids. As used in this disclosure the term antigen comprehends such macromolecules as well. Moreover, a nucleic acid based vaccine may encode an enzyme or enzymes necessary to the synthesis of such a macromolecule and thereby confer antigen expression on an APC.

A powerful method has been recently developed for identifying new peptides that are useful in the invention. Genes determined to express protein with high exclusivity in tumor cells or microbial cells (e.g. viruses) can be identified using a so called SEREX process, which involves expression cloning using tumor cell libraries and screening these libraries against immunoglobulin in patient sera. Over one hundred genes have recently been identified from tumor biopsies using this process. These genes can now be used in a peptide prediction algorithm developed by Hans-Georg Rammensee. Algorithms have been developed for all major HLA types found in the human population. First the protein sequence is “translated” based on the gene sequence. The algorithms can predict peptide epitopes for various HLA types based on the protein sequence. Since the predicted peptides are indeed predictions and are not always naturally found on cells, tumor samples are used to confirm the predicted peptides by actually isolating minute trace peptide from tumors. Being able to calculate the exact mass of the predicted peptides allows trace peptide identification using ultrasensitive mass spectrophotometry, which can detect peptides in quantities less that that which would permit peptide sequencing and identification. Once these tumor-associated peptides have been identified they are suitable for use in the invention, since peptides of a known sequence may be synthesized in large quantities (several grams) providing for sufficient amounts of peptides for use in this invention.

In addition to the imperfection of existing prediction algorithms for MHC binding, some peptides that would be fully capable of binding to MHC may never be liberated by protoelytic processing from the complete protein antigen. Methods for evaluating which peptides will be liberated by proteasomal processing have been developed, e.g. U.S. patent application Ser. No. 09/561,074 supra, increasing the efficiency with which useful epitopes can be discovered. Moreover, proteasomal processing can differ between target cell and APC such that care must be taken in the identification and selection of epitopes and in vaccine design so that the vaccine will induce a response that will in fact recognize the target cell. These issues are more fully discussed in U.S. patent application Ser. No. 09/560,465 entitled “EPITOPE SYNCHRONIZATION,” which is incorporated herein by reference in its entirety.

Thus it can be seen that another aspect of this invention is a process for preparing a composition useful in a device of this invention as discussed hereinafter. The process comprises identifying a gene determined to express a protein with high exclusivity in a tumor or microbial cell, cloning cell libraries, screening the libraries against immunoglobulin in patient sera, using the algorithm defined in the literature developed by Hans-George Rammensee to predict an epitope for the HLA type protein based on the gene sequence, matching the predicted antigen sequence to a patient tumor sample, isolating the matched antigen, and preparing a composition of the antigen for use in a delivery device as discussed hereinafter.

Examples of large, protein-based antigens include the following:

Differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS. These protein-based antigens are known and available to those of skill in the art in the literature or commercially.

Examples of peptide antigens of 8-15 amino acids include those set forth in Table I, Table II, and Table III. Table I sets forth antigens that are virally derived. The Table shows the virus type, the protein expressed by the virus, the amino acid (AA) position on the viral protein, the AA sequence of the T-cell epitope/MHC ligand, the type of MHC molecule presenting the antigen, and a reference source. A more complete list is provided in the book by Han-Georg Rammensee, Jutta Bachmann, and Stefan Stevanovic entitled “MHC Ligands and Peptide Motifs,” Springer-Verlag, Germany, 1997 Landes Bioscience, Austin, Tex.). The reference number given in Table I is the same number (and reference source) given in Table 5.3 of the above Rammensee book, all of which is incorporated herein by reference.

TABLE I Viral epitopes on MHC class 1 molecules AA T cell epitope MHC Virus Protein Position ligand (Antigen) MHC molecule Ref. Adenovirus 3 E3 9Kd   30-38 LIVIGILIL HLA-A*0201 104 (SEQ. ID NO.: 1) Adenovirus 5 EIA  234-243 SGPSNTPPEI H2-Db 105 (SEQ. ID NO.: 2) Adenovirus 5 EIB  192-200 VNIRNCCYI H2-Db 106 (SEQ. ID NO.: 3) Adenovirus 5 EIA  234-243 SGPSNIPPEI (T > I) H2-Db 106 (SEQ. ID NO.: 4) CSFV NS 2276-2284 ENALLVALF SLA, haplotype 107 polyprotein (SEQ. ID NO.: 5 d/d Dengue virus NS3  500-508 TPEGIIPTL HLA-B*3501 108, 109 4 (SEQ. ID NO.: 6 EBV LMP-2  426-434 CLGGLLTMV HLA-A*0201 110 (SEQ. ID NO.: 7) EBV EBNA-1  480-484 NIAEGLRAL HLA-A*0201 111 (SEQ. ID NO.: 8) EBV EBNA-1  519-527 NLRRGTALA HLA-A*0201 111 (SEQ. ID NO.: 9) EBV EBNA-1  525-533 ALAIPQCRL HLA-A*0201 111 (SEQ. ID NO.: 10) EBV EBNA-1  575-582 VLKDAIKDL HLA-A*0201 111 (SEQ. ID NO.: 11) EBV EBNA-1  562-570 FMVFLQTHI HLA-A*0201 111 (SEQ. ID NO.: 12) EBV EBNA-2   15-23 HLIVDTDSL HLA-A*0201 111 (SEQ. ID NO.: 13) EBV EBNA-2   22-30 SLGNPSLSV HLA-A*0201 111 (SEQ. ID NO.: 14) EBV EBNA-2  126-134 PLASAMRML HLA-A*0201 111 (SEQ. ID NO.: 15) EBV EBNA-2  132-140 RMLWMANYI HLA-A*0201 111 (SEQ. ID NO.: 16) EBV EBNA-2  133-141 MLWMANYIV HL.A-A*0201 111 (SEQ. ID NO.: 17) EBV EBNA-2  151-159 ILPQGPQTA HLA-A*0201 111 (SEQ. ID NO.: 18) EBV EBNA-2  171-179 PLRPTAPTI HLA-A*0201 111 (SEQ. ID NO.: 19) EBV EBNA-2  205-213 PLPPATLTV HLA-A*0201 111 (SEQ. ID NO.: 20) EBV EBNA-2  246-254 RMHLPVLHV HLA-A*0201 111 (SEQ, ID NO.: 21) EBV EBNA-2  287-295 PMPLPPSQL HLA-A*0201 111 (SEQ, ID NO.: 22) EBV EBNA-2  294-302 QLPPPAAPA HLA-A*0201 111 (SEQ, ID NO.: 23) EBV EBNA-2  381-389 SMPELSPVL HLA-A*0201 111 (SEQ. ID NO.: 24) EBV EBNA-2  453-461 DLDESWDYI HLA-A*0201 111 (SEQ. ID NO.: 25) EBV BZLF1   43-51 PLPCVLWPV HLA-A*0201 111 (SEQ. ID NO.: 26) EBV BZLF1  167-175 SLEECDSEL HLA-A*0201 111 (SEQ. ID NO.: 27) EBV BZLF1  176-184 EIKRYKNRV HLA-A*0201 111 (SEQ. ID NO.: 28) EBV BZLF1  195-203 QLLQHYREV HLA-A*0201 111 (SEQ. ID NO.: 29) EBV BZLF1  196-204 LLQHYREVA HLA-A*0201 111 (SEQ. ID NO.: 30) EBV BZLF1  217-225 LLKQMCPSL HLA-A*0201 111 (SEQ. ID NO.: 31) EBV BZLF1  229-237 SIIPRTPDV HLA-A*0201 111 (SEQ. ID NO.: 32) EBV EBNA-6  284-293 LLDFVRFMGV HLA-A*0201 112 (SEQ. ID NO.: 33) EBV EBNA-3  464-472 SVRDRLARL HLA-A*0203 113 (SEQ. ID NO.: 34) EBV EBNA-4  416-424 IVTDFSVIK HLA-A*1101 114, 115 (SEQ. ID NO.: 35) EBV EBNA-4  399-408 AVFDRKSDAK HLA-A*0201 116 (SEQ. ID NO.: 36) EBV EBNA-3  246-253 RYSIFFDY HLA-A24 113 (SEQ. ID NO.: 37) EBV EBNA-6  881-889 QPRAPIRPI HLA-B7 117 (SEQ. ID NO.: 38) EBV EBNA-3  379-387 RPPIFIRRI. HLA-B7 117 (SEQ. ID NO.: 39) EBV EBNA-1  426-434 EPDVPPGAI HLA-B7 111 (SEQ. ID NO.: 40) EBV EBNA-1  228-236 IPQCRLTPL HLA-B7 111 (SEQ. ID NO.: 41) EBV EBNA-1  546-554 GPGPQPGPL HLA-B7 111 (SEQ. ID NO.: 42) EBV EBNA-1  550-558 QPGPLRESI HLA-B7 111 (SEQ. ID NO.: 43) EBV EBNA-1   72-80 R.PQKRPSCI HLA-B7 111 (SEQ. ID NO.: 44) EBV EBNA-2  224-232 PPTPLLTVL HLA-B7 111 (SEQ. ID NO.: 45) EBV EBNA-2  241-249 TPSPPRMHL HLA-B7 111 (SEQ. ID NO.: 46) EBV EBNA-2  244-252 PPRMHLPVL HLA-B7 111 (SEQ. ID NO.: 47) EBV EBNA-2  254-262 VPDQSMHPL HLA-B7 111 (SEQ. ID NO.: 48) EBV EBNA-2  446-454 PPSIDPADL HLA-B7 111 (SEQ. ID NO.: 49) EBV BZLFI   44-52 LPCVLWPVL HLA-B7 111 (SEQ. ID NO.: 50) EBV BZLFI  222-231 CPSLDVDSII HLA-B7 111 (SEQ. ID NO.: 51) EBV BZLFI  234-242 TPDVLHEDL HLA-B7 111 (SEQ. ID NO.: 52) EBV EBNA-3  339-347 FLRGRAYGL HLA-B8 118 (SEQ. ID NO.: 53) EBV EBNA-3   26-34 QAKWRLQTL HLA-B8 113 (SEQ. ID NO.: 54) EBV EBNA-3  325-333 AYPLHEQHG HLA-B8 116 (SEQ. ID NO.: 55) EBV EBNA-3  158-166 YIKSFVSDA HLA-B8 116 (SEQ. ID NO.: 56) EBV LMP-2  236-244 RRRWRRLTV HLA-B*2704 119 (SEQ. ID NO.: 57) EBV EBNA-6  258-266 RRIYDLIEL HLA-B*2705 119 (SEQ. ID NO.: 58) EBV EBNA-3  458-466 YPLHEQHGM HLA-B*3501 120 (SEQ. ID NO.: 59) EBV EBNA-3  458-466 YPLHEQHGM HLA-B*3503 113 (SEQ. ID NO.: 59) HCV NS3  389-397 HSKKKCDEL HLA-B8 145 (SEQ. ID NO.: 60) HCV env E   44-51 ASRCWVAM HLA-B*3501 146 (SEQ. ID NO.: 61) HCV core   27-35 GQIVGGVYL HLA-B*40012 147 protein (SEQ. ID NO.: 62) HCV NSI   77-85 PPLTDFDQGW HLA-B*5301 145 (SEQ. ID NO.: 63) HCV core   18-27 LMGYIPLVGA H2-Dd 138 protein (SEQ. ID NO.: 64) HCV core   16-25 ADLMGYIPLV H2-Dd 148 protein (SEQ. ID NO.: 65) HCV NS5  409-424 MSYSWTGALVTPC H2-Dd 149 AEE (SEQ. ID NO.: 66) HCV NS1  205-213 KHPDATYSR Papa-A06 150 (SEQ. ID NO.: 67) HCV-1 NS3  400-409 KLVALGINAV HLA-A*0201 141 (SEQ. ID NO.: 68) HCV-1 NS3  440-448 GDFDSVIDC Patr-B16 151 (SEQ. ID NO.: 69) HCV-1 env E  118-126 GNASRCWVA Patr-B16 151 (SEQ. ID NO.: 70) HCV-1 NSI  159-167 TRPPLGNWF Patr-B13 151 (SEQ. ID NO.: 71) HCV-1 NS3  351-359 VPHPNIEEV Patr-B13 151 (SEQ. ID NO.: 72) HCV-1 NS3  438-446 YTGDFDSVI Patr-B01 151 (SEQ. ID NO.: 73) HCV-1 NS4  328-335 SWAIKWEY Patr-A11 151 (SEQ. ID NO.: 74) HCV-1 NSI  205-213 KHPDATYSR Patr-A04 150 (SEQ. ID NO.: 75) HCV-1 NS3  440-448 GDFDSVIDC Patr-A04 150 (SEQ. ID NO.: 76) HIV gp4l  583-591 RYLKDQQLL HLA_A24 152 (SEQ. ID NO.: 77) HIV gagp24  267-275 IVGLNKIVR HLA-A*3302 153, 154 (SEQ. ID NO.: 78) HIV gagp24  262-270 EIYKRWIIL HLA-B8 155, 156 (SEQ. ID NO.: 79) HIV gagp24  261-269 GEIYKRWII HLA-B8 155, 156 (SEQ. ID NO.: 80) HIV gagp17   93-101 EIKDTKEAL HLA-B8 155, 157 (SEQ. ID NO.: 81) HIV gp41  586-593 YLKDQQLL HLA-B8 158 (SEQ. ID NO.: 82) HIV gagp24  267-277 ILGLNKIVRMY HLA-B* 1501 153 (SEQ. ID NO.: 83) HIV gp41  584-592 ERYLKDQQL HLA-B14 158 (SEQ. ID NO.: 84) HIV nef  115-125 YHTQGYFPQWQ HLA-B17 159 (SEQ. ID NO.: 85) HIV nef  117-128 TQGYFPQWQNYT HLA-B17 159 (SEQ. ID NO.: 86) HIV gp120  314-322 GRAFVTIGK HLA-B*2705 160, 184 (SEQ. ID NO.: 87) HIV gagp24  263-271 KRWIILGLN HLA-B*2702 161 (SEQ. ID NO.: 88) HIV nef   72-82 QVPLRPMTYK HLA-B*3501 159 (SEQ. ID NO.: 89) HIV nef  117-125 TQGYFPQWQ HLA-B*3701 159 (SEQ. ID NO.: 90) HIV gagp24  143-151 HQAISPRTI, HLA-Cw*0301 162 (SEQ. ID NO.: 91) HIV gagp24  140-151 QMVHQAISPRTL HLA-Cw*0301 162 (SEQ. ID NO.: 92) HIV gp120  431-440 MYAPPIGGQI H2-Kd 163 (SEQ. ID NO.: 93) HIV gp160  318-327 RGPGRAFVTI H2-Dd 164, 165 (SEQ. ID NO.: 94) HIV gp120   17-29 MPGRAFVTI H2-Ld 166, 167 (SEQ. ID NO.: 95) HIV-1 RT  476-484 ILKEPVHGV HLA-A*0201 168, 169 (SEQ. ID NO.: 96) HIV-1 nef  190-198 AFHHVAREL HLA-A*0201 170 (SEQ ID NO.: 97) HIV-1 gp160  120-128 KLTPLCVTL HLA-A*0201 171 (SEQ ID NO.: 98) HIV-1 gp]60  814-823 SLLNATDIAV HLA-A*0201 171 (SEQ ID NO.: 99) HIV-1 RT  179-187 VIYQYMDDL HLA-A*0201 172 (SEQ ID NO.: 100) HIV-1 gagp 17   77-85 SLYNTVATL HLA-A*0201 173 (SEQ ID NO.: 101) HIV-1 gp160  315-329 RGPGRAFVTI HLA-A*0201 174 (SEQ ID NO.: 102) HIV-1 gp41  768-778 RLRDLLLIVTR HLA-A3 175, 178 (SEQ ID NO.: 103) HIV-1 nef   73-82 QVPLRPMTYK HLA-A3 176 (SEQ ID NO.: 104) HIV-1 gp120   36-45 TVYYGVPVWK HLA-A3 177 (SEQ. ID NO.: 105) HIV-1 gagp17   20-29 RLRPGGKKK HLA-A3 177 (SEQ. ID NO.: 106) HIV-1 gp120   38-46 VYYGVPVWK HLA-A3 179 (SEQ. ID NO.: 107) HIV-1 nef   74-82 VPLRPMTYK HLA-a*1101 114 (SEQ. ID NO.: 108) HIV-1 gagp24  325-333 AIFQSSMTK HLA-A*1101 114 (SEQ. ID NO.: 109) HIV-1 nef   73-82 QVPLRPMTYK HLA-A*1101 180 (SEQ. ID NO.: 104) HIV-1 nef   83-94 AAVDLSHFLKEK HLA-A*1101 159 (SEQ, ID NO.: 110) HIV-1 gagp24  349-359 ACQGVGGPGGHK HLA-A*1101 181 (SEQ. ID NO.: 111) HIV-1 gagp24  203-212 ETINEEAAEW HLA-A25 182 (SEQ. ID NO.: 112) HIV-1 nef  128-137 TPGPGVRYPL HLA-B7 159 (SEQ. ID NO.: 113) HIV-1 gagp 17   24-31 GGKKKYKL HLA-B8 183 (SEQ. ID NO.: 114) HIV-1 gp120    2-10 RVKEKYQHL HLA-B8 181 (SEQ. ID NO.: 115) HIV-1 gagp24  298-306 DRFYKTLRA HLA-B 14 173 (SEQ. ID NO.: 116) HIV-1 NEF  132-147 GVRYPLTFGWCYK HLA-B18 159 LVP (SEQ. ID NO.: 117) HIV-1 gagp24  265-24 KRWIILGLNK HLA-B*2705 184, 153 (SEQ. ID NO.: 118) HIV-1 nef  190-198 AFHHVAREL HLA-B*5201 170 (SEQ. ID NO.: 97) EBV EBNA-6  335-343 KEHVIQNAF HLA-B44 121 (SEQ. ID NO.: 119) EBV EBNA-6  130-139 EENLLDFVRF HLA-B*4403 122 (SEQ. ID NO.: 120) EBV EBNA-2   42-51 DTPLIPLTIF HLA-B51 121 (SEQ. ID NO.: 121) EBV EBNA-6  213-222 QNGALAINTF HLA-1362 112 (SEQ. ID NO.: 122) EBV EBNA-3  603-611 RLRAEAGVK HLA-A3 123 (SEQ. ID NO.: 123) HBV sAg  348-357 GLSPTVWLSV HLA-A*0201 124 (SEQ. ID NO.: 124) HBV SAg  335-343 WLSLLVPFV HLA-A*0201 124 (SEQ. ID NO.: 125) HBV cAg   18-27 FLPSDFFPSV HLA-A*0201 125, 126, (SEQ. ID NO.: 126) 127 HBV cAg   18-27 FLPSDFFPSV HLA-A*0202 127 (SEQ. ID NO.: 126) HBV cAg   18-27 FLPSDFFPSV HLA-A*0205 127 (SEQ. ID NO.: 126) HBV cAg   18-27 FLPSDFFPSV HLA-A*0206 127 (SEQ. ID NO.: 126) HBV pol  575-583 FLLSLGIHL HLA-A*0201 128 (SEQ. ID NO.: 127) HBV pol  816-824 SLYADSPSV HLA-A*0201 128 (SEQ. ID NO.: 128) HBV pol  455-463 GLSRYVARL HLA-A*0201 128 (SEQ. ID NO.: 129) HBV env  338-347 LLVPFVQWFV HLA-A*0201 129 (SEQ. ID NO.: 130) HBV pol  642-650 ALMPLYACI HLA-A*0201 129 (SEQ. ID NO.: 131) HBV env  378-387 LLPIFFCLWV HLA-A*0201 129 (SEQ. ID NO.: 132) HBV pol  538-546 YMDDVVLGA HLA-A*0201 129 (SEQ. ID NO.: 133) HBV env  250-258 LLLCLIFLL HLA-A*0201 130 (SEQ. ID NO.: 134) HBV env  260-269 LLDYQGMLPV HLA-A*0201 130 (SEQ. ID NO.: 135) HBV env  370-379 SIVSPFIPLL HLA-A*0201 130 (SEQ. ID NO.: 136) HBV env  183-191 FLLTRILTI HLA-A*0201 130 (SEQ. ID NO.: 137) HBV cAg   88-96 YVNVNMGLK HLA-A* 1101 131 (SEQ. ID NO.: 138) HBV cAg  141-151 STLPETTVVRR HLA-A*3101 132 (SEQ. ID NO.: 139) HBV cAg  141-151 STLPETTVVRR HLA-A*6801 132 (SEQ. ID NO.: 139) HBV cAg   18-27 FLPSDFFPSV HLA-A*6801 127 (SEQ. ID NO.: 126) HBV sAg   28-39 IPQSLDSWWTSL H2-Ld 133 (SEQ. ID NO.: 140) HBV cAg   93-100 MGLKFRQL H2-Kb 134 (SEQ. ID NO.: 141) HBV preS  141-149 STBXQSGXQ HLA-A*0201 135 (SEQ. ID NO.: 142) HCMV gp B  618-628 FIAGNSAYEYV HLA-A*0201 124 (SEQ. ID NO.: 143) HCMV E1  978-989 SDEEFAIVAYTL HLA-B18 136 (SEQ. ID NO.: 144) HCMV pp65  397-411 DDVWTSGSDSDEE HLA-b35 137 LV (SEQ. ID NO.: 145) HCMV pp65  123-131 IPSINVHHY HLA-B*3501 136 (SEQ. ID NO.: 146) HCMV pp65  495-504 NLVPMVATVO HLA-A*0201 137 (SEQ. ID NO.: 147) HCMV pp65  415-429 RKTPRVTOGGAMA HLA-B7 137 GA (SEQ. ID NO.: 148) HCV MP   17-25 DLMGYIPLV HLA-A*0201 138 (SEQ. ID NO.: 149) HCV MP   63-72 LLALLSCLTV HLA-A*0201 139 (SEQ. ID NO.: 150) HCV MP  105-112 ILHTPGCV HLA-A*0201 139 (SEQ. ID NO.: 151) HCV env E   66-75 QLRRHIDLLV HLA-A*0201 139 (SEQ. ID NO.: 152) HCV env E   88-96 DLCGSVFLV HLA-A*0201 139 (SEQ. ID NO.: 153) HCV env E  172-180 SMVGNWAKV HLA-A*0201 139 (SEQ. ID NO.: 154) HCV NSI  308-316 HLIIQNIVDV HLA-A*0201 139 (SEQ. ID NO.: 155) HCV NSI  340-348 FLLLADARV HLA-A*0201 139 (SEQ. ID NO.: 156) HCV NS2  234-246 GLRDLAVAVEPVV HLA-A*0201 139 (SEQ. ID NO.: 157) HCV NSI   18-28 SLLAPGAKQNV HLA-A*0201 139 (SEQ. ID NO.: 158) HCV NSI   19-28 LLAPGAKQNV HLA-A*0201 139 (SEQ. ID NO.: 159) HCV NS4  192-201 LLFNILGGWV HLA-A*0201 129 (SEQ. ID NO.: 160) HCV NS3  579-587 YLVAYQATV HLA-A*0201 129 (SEQ. ID NO.: 161) HCV core   34-43 YLLPRRGPRL HLA-A*0201 129 protein (SEQ. ID NO.: 162) HCV MP   63-72 LLALLSCLTI HLA-A*0201 129 (SEQ. ID NO.: 163) HCV NS4  174-182 SLMAFTAAV HLA-A*0201 140 (SEQ. ID NO.: 164) HCV NS3   67-75 CINGVCWTV HLA-A*0201 140 (SEQ. ID NO.: 165) HCV NS3  163-171 LLCPAGHAV HLA-A*0201 141 (SEQ. ID NO.: 166) HCV NS5  239-247 ILDSFDPLV HLA-A*0201 141 (SEQ. ID NO.: 167) HCV NS4A  236-244 ILAGYGAGV HLA-A*0201 142 (SEQ. ID NO.: 168) HCV NS5  714-722 GLQDCTMLV HLA-A*0201 142 (SEQ. ID NO.: 169) HCV NS3  281-290 TGAPVTYSTY HLA-A*0201 143 (SEQ. ID NO.: 170) HCV NS4A  149-157 HMWNFISGI HLA-A*0201 144 (SEQ. ID NO.: 171) HCV NS5  575-583 RVCEKMALY HLA-A*0201-A3 145 (SEQ. ID NO.: 172) HCV NS1  238-246 TINYTIFK HLA-A*1101 145 (SEQ. ID NO.: 173) HCV NS2  109-116 YISWCLWW HLA-A23 145 (SEQ. ID NO.: 174) HCV core   40-48 GPRLGVRAT HLA-B7 145 protein (SEQ. ID NO.: 175) HIV-1 gp120  380-388 SFNCGGEFF HLA-Cw*0401 185 (SEQ. ID NO.: 176) HIV-1 RT  206-214 TEMEKEGKI H2-Kk 186 (SEQ. ID NO.: 177) HIV-1 p17   18-26 KIRLRPGGK HLA-A*0301 187 (SEQ. ID NO.: 178) HIV-1 P17   20-29 RLRPGGKKKY HLA-A*0301 188 (SEQ. ID NO.: 179) HIV- 1 RT  325-333 AIFQSSMTK HLA-A*0301 188 (SEQ..ID NO.: 180) HIV-1 p17   84-92 TLYCVHQRI HLA-A11 188 (SEQ. ID NO.: 181) HIV-1 RT  508-517 IYQEPFKNLK HLA-A11 188 (SEQ. ID NO.: 182) HIV-1 p17   28-36 KYKLKHIVW HLA-A24 188 (SEQ. ID NO.: 183) HIV-1 gp120   53-62 LFCASDAKAY HLA-A24 189 (SEQ. ID NO.: 184) HIV-1 gagp24  145-155 QAISPRTLNAW HLA-A25 188 (SEQ. ID NO.: 185) HIV-1 gagp24  167-175 EVIPMFSAL FILA-A26 188 (SEQ. ID NO.: 186) HIV-1 RT  593-603 ETFYVDGAANR HLA-A26 188 (SEQ. ID NO.: 187) HIV-1 gp41  775-785 RLRDLLLIVTR HLA-A31 190 (SEQ. ID NO.: 188) HIV-1 RT  559-568 PIQKETWETW HLA-A32 187 (SEQ. ID NO.: 189) HIV-1 gp120  419-427 RIKQIINMW HLA-A32 187 (SEQ. ID NO.: 190) HIV-1 RT   71-79 ITLWQRPLV HLA-A*6802 188 (SEQ. ID NO.: 191) HIV-1 RT   85-93 DTVLEEMNL HLA-A*6802 188 (SEQ. ID NO.: 192) HIV-1 RT   71-79 ITLWQRPLV HLA-A*7401 188 (SEQ. ID NO.: 193) HIV-1 gag p24  148-156 SPRTLNAWV HLA-B7 188 (SEQ. ID NO.: 194) HIV-1 gagp24  179-187 ATPQDLNTM HLA-B7 188 (SEQ. ID NO.: 195) HIV-1 gp120  303-312 RPNNNTRKSI FILA-B7 188 (SEQ. ID NO.: 196) HIV-1 gp41  843-851 IPRRIRQGL HLA-B7 188 (SEQ. ID NO.: 197) HIV-1 p17   74-82 ELRSLYNTV HLA-B8 188 (SEQ. ID NO.: 198) HIV-1 nef   13-20 WPTVRERM HLA-B8 188 (SEQ. ID NO.: 199) HIV-1 nef   90-97 FLKEKGGL HLA-B8 188 (SEQ. ID NO.: 200) HIV-1 gag p24  183-191 DLNTMLNTV HLA-B14 191 (SEQ. ID NO.: 568) HIV-1 P17   18-27 KIRLRPGGKK HLA-B27 188 (SEQ. ID NO.: 201) HIV-1 pl7   19-27 IRLRPGGKK HLA-B27 188 (SEQ. ID NO.: 202) HIV-1 gp41  791-799 GRRGWEALKY HLA-B27 188 (SEQ. ID NO.: 203) HIV-1 nef   73-82 QVPLRPMTYK HLA-B27 188 (SEQ. ID NO.: 204) HIV-1 GP41  590-597 RYLKDQQL HLA-B27 192 (SEQ. ID NO.: 205) HIV-1 nef  105-114 RRQDILDLWI HLA-B*2705 188 (SEQ. ID NO.: 206) HIV-1 nef  134-141 RYPLTFGW HLA-B*2705 188 (SEQ. ID NO.: 207) HIV-1 p17   36-44 WASRELERF HLA-B35 188 (SEQ. ID NO.: 208) HIV-1 GAG  262-270 TVLDVGDAY HLA-B35 188 P24 (SEQ. ID NO.: 209) HIV-1 gp120   42-52 VPVWKEATTTL HLA-B35 188 (SEQ. ID NO.: 210) HIV-1 P17   36-44 NSSKVSQNY HLA-B35 193 (SEQ. ID NO.: 221) HIV-1 gag p24  254-262 PPIPVGDIY HLA-B35 193 (SEQ. ID NO.: 212) HIV-1 RT  342-350 HPDIVIYQY HLA-B35 193 (SEQ. ID NO.: 213) HIV-1 gp41  611-619 TAVPWNASW HLA-B35 194 (SEQ. ID NO.: 214) HIV-1 gag  245-253 NPVPVGNIY HLA-B35 193 (SEQ. ID NO.: 215) HIV-1 nef  120-128 YFPDWQNYT HLA-B37 188 (SEQ. ID NO.: 216) HIV-1 gag p24  193-201 GHQAAMQML HLA-B42 188 (SEQ. ID NO.: 217) HIV-1 p17   20-29 RLRPGGKKKY HLA-B42 188 (SEQ. ID NO.: 218) HIV-1 RT  438-446 YPGIKVRQL HLA-B42 188 (SEQ. ID NO.: 219) HIV-1 RT  591-600 GAETFYVDGA HLA-B45 188 (SEQ. ID NO.: 220) HIV-1 gag p24  325-333 NANPDCKTI HLA-B51 188 (SEQ. ID NO.: 221) HIV-1 gag p24  275-282 RMYSPTSI HLA-B52 188 (SEQ. ID NO.: 222) HIV-1 gp120   42-51 VPVWKEATTT HLA-B*5501 192 (SEQ. ID NO.: 223) HIV-1 gag p24  147-155 ISPRTLNAW HLA-B57 188 (SEQ. ID NO.: 224) HIV-1 gag p24  240-249 TSTLQEQIGW HLA-B57 188 (SEQ. ID NO.: 225) HIV-1 gag p24  162-172 KAFSPEVIPMF HLA-B57 188 (SEQ. ID NO.: 226) HIV-1 gag p24  311-319 QASQEVKNW HLA-B57 188 (SEQ. ID NO.: 227) HIV-1 gag p24  311-319 QASQDVKNW HLA-B57 188 (SEQ. ID NO.: 228) HIV-1 nef  116-125 HTQGYFPDWQ HLA-B57 188 (SEQ. ID NO.: 229) HIV-1 nef  120-128 YFPDWQNYT HLA-B57 188 (SEQ. ID NO.: 230) HIV-1 gag p24  240-249 TSTLQEQIGW HLA-B58 188 (SEQ. ID NO.: 231) HIV-1 p17   20-29 RLRPGGKKKY HLA-B62 188 (SEQ. ID NO.: 232) HIV-1 p24  268-277 LGLNKJVRMY HLA-B62 188 (SEQ. ID NO.: 233) HIV-1 RT  415-426 LVGKLNWASQIY HLA-B62 188 (SEQ. ID NO.: 234) HIV-1 RT  476-485 ILKEPVHGVY HLA-B62 188 (SEQ. ID NO.: 235) HIV-1 nef  117-127 TQGYFPDWQNY HLA-B62 188 (SEQ. ID NO.: 236) HIV-1 nef   84-91 AVDLSHFL HLA-B62 188 (SEQ. ID NO.: 237) HIV-1 gag p24  168-175 VIPMFSAL HLA-Cw*0102 188 (SEQ. ID NO.: 238) HIV-1 gp120  376-384 FNCGGEFFY HLA-A29 196 (SEQ. ID NO.: 239) HIV-1 gp120  375-383 SFNCGGEFF HLA-B15 196 (SEQ. ID NO.: 240) HIV-1 nef  136-145 PLTFGWCYKL HLA-A*0201 197 (SEQ. ID NO.: 241) HIV-1 nef  180-189 VLEWRFDSRL HLA-A*0201 197 (SEQ. ID NO.: 242) HIV-1 nef   68-77 FPVTPQVPLR HLA-B7 197 (SEQ. ID NO.: 243) HIV-1 nef  128-137 TPGPGVRYPL HLA-B7 197 (SEQ. ID NO.: 244) HIV-1  gag p24  308-316 QASQEVKNW HLA-Cw*0401 521 (SEQ. ID NO.: 245) HIV-1 IIIB RT  273-282 VPLDEDFRKY HLA-B35 181 (SEQ. ID NO.: 246) HIV-1 IIIB RT   25-33 NPDIVIYQY HLA-B35 181 (SEQ. ID NO.: 247) HIV-1 IIIB gp41  557-565 RAIEAQAHL HLA-B51 181 (SEQ. ID NO.: 248) HIV-1 IIIB RT  231-238 TAFTIPSI HLA-B51 181 (SEQ. ID NO.: 249) HIV-1 IIIB p24  215-223 VHPVHAGPIA HLA-B*5501 181 (SEQ. ID NO.: 250) HIV-1 IIIB gp120  156-165 NCSFNISTSI HLA-Cw8 181 (SEQ. ID NO.: 251) HIV-1 IIIB  gp120  241-249 CTNVSTVQC HLA-Cw8 181 (SEQ. ID NO.: 252) HIV-1 5F2 gp120  312-320 IGPGRAFHT H2-Dd 198 (SEQ. ID NO.: 253) HIV-1 5F2 pol   25-33 NPDIVIYQY HLA-B*3501 199 (SEQ. ID NO.: 254) HIV-1 5F2 pot  432-441 EPIVGAETFY HLA-B*3501 199 (SEQ. ID NO.: 255) HIV-1 5F2 pol  432-440 EPIVGAETF HLA-B*3501 199 (SEQ. ID NO.: 256) HIV-1 5F2 pol    6-14 SPAIFQSSM HLA-B*3501 199 (SEQ. ID NO.: 257) HIV-1 5F2 pol   59-68 VPLDKDFRKY HLA-B*3501 199 (SEQ. ID NO.: 258) HIV-1 5F2 po1    6-14 IPLTEEAEL HLA-B*3501 199 (SEQ. ID NO.: 259) HIV-1 5F2 nef   69-79 RPQVPLRPMTY HLA-B*3501 199 (SEQ. ID NO.: 260) HIV-1 5F2 nef   66-74 FPVRPQVPL HLA-B*3501 199 (SEQ. ID NO.: 261) HIV-1 5F2 env   10-18 DPNPQEVVL HLA-B*3501 199   (SEQ. ID NO.: 262) HIV-1 5F2 env    7-15 RPIVSTQLL HLA-B*3501 199 (SEQ. ID NO.: 263) HIV-1 5F2 pol    6-14 IPLTEEAEL HLA-B51 199 (SEQ. ID NO.: 264) HIV-1 5F2 env   10-18 DPNPQEVVL HLA-B51 199 (SEQ. ID NO.: 265) HIV-1 5F2 gagp24  199-207 AMQMLKETI H2-Kd 198 (SEQ. ID NO.: 266) HIV-2 gagp24  182-190 TPYDrNQML HLA-B*5301 200 (SEQ. ID NO.: 267) HIV-2 gag  260-269 RRWIQLGLQKV HLA-B*2703 188 (SEQ. ID NO.: 268) HIV-1 5F2 gp4l  593-607 GIWGCSGKLICTTA HLA-B17 201 V (SEQ. ID NO.: 269) HIV-1 5F2 gp41  753-767  ALIWEDLRSLCLFS HLA-B22 201 Y (SEQ. ID NO.: 270) HPV 6b E7   21-30 GLHCYEQLV HLA-A*0201 202 (SEQ. ID NO.: 271) HPV 6b E7   47-55 PLKQHFQIV HLA-A*0201 202 (SEQ. ID NO.: 272) HPV11 E7    4-12 RLVTLKDIV HLA-A*0201 202 (SEQ. ID NO.: 273) HPV16 E7   86-94 TLGIVCPIC HLA-A*0201 129 (SEQ. ID NO.: 274) HPV16 E7   85-93 GTLGIVCPI HLA-A*0201 129 (SEQ. ID NO.: 275) HPV16 E7   12-20 MLDLQPETT HLA-A*0201 129 (SEQ. ID NO.: 276) HPV16 E7   11-20 YMLDLQPETT HLA-A*0201 203 (SEQ. ID NO.: 277) HPV16 E6   15-22 RPRKLPQL HLA-B7 204 (SEQ. ID NO.: 278) HPV16 E6   49-57 RAHYNIVTF HW-Db 205 (SEQ. ID NO.: 279) HSV gp B  498-505 SSIEFARL H2-Kb 206 (SEQ. ID NO.: 280) HSV-1 gp C  480-488 GIGIGVLAA HLA-A*0201 104 (SEQ. ID NO.: 281) HSV-1 ICP27  448-456 DYATLGVGV H2-Kd 207 (SEQ. ID NO.: 282) HSV-1 ICP27  322-332 LYRTFAGNPRA H2-Kd 207 (SEQ. ID NO.: 283) HSV-1 UL39  822-829 QTFDFGRL H2-Kb 208 (SEQ. ID NO.: 284) HSV-2 gpC  446-454 GAGIGVAVL HLA-A*0201 104 (SEQ. ID NO.: 285) HLTV-1 TAX   11-19 LLFGYPVYV HLA-A*0201 209 (SEQ. ID NO.: 286) Influenza MP   58-66 GILGFVFTL HLA-A*0201  68, 169, 2 (SEQ. ID NO.: 287)  09, 210, 2  11 Influenza MP   59-68 ILGFVFTLTV HLA-A*0201 168, 212, (SEQ. ID NO.: 288) 213 Influenza NP  265-273 ILRGSVAHK HLA-A3 214 (SEQ. ID NO.: 289) Influenza NP   91-99 KTGGPIYKR HLA-A*6801 215, 216 (SEQ. ID NO.: 290) Influenza NP  380-388 ELRSRYWAI HLA-B8 217 (SEQ. ID NO.: 291) Influenza NP  381-388 LRSRYWAI HLA-B*2702 218 (SEQ. ID NO.: 292) Influenza NP  339-347 EDLRVLSFI HLA-B*3701 219 (SEQ. ID NO.: 293) Influenza NSI  158-166 GEISPLPSL HLA-B44 220 (SEQ. ID NO.: 294) Influenza NP  338-346 FEDLRVLSF HLA-B44 220 (SEQ. ID NO.: 295) Influenza NSI  158-166 GEISPLPSL HLA-B*4402 220 (SEQ. ID NO.: 294) Influenza NP  338-346 FEDLRVLSF HLA-B*4402 220 (SEQ. ID NO.: 295) Influenza PBI  591-599 VSDGGPKLY HLA-A1 214, 29 (SEQ. ID NO.: 296) Influenza A NP   44-52 CTELKLSDY HLA-A1  29 (SEQ. ID NO.: 297) Influenza NSI  122-130 AIMDKNIIL HLA-A*0201 221 (SEQ. ID NO.: 298) Influenza A NSI  123-132 IMDKNIILKA HLA-A*0201 221 (SEQ. ID NO.: 299) Influenza A NP  383-391 SRYWAIRTR HLA-B*2705 160, 184 (SEQ. ID NO.: 300) Influenza A NP  147-155 TYQRTRALV H2-Kd 222, 223 (SEQ. ID NO.: 301) Influenza A HA  210-219 TYVSVSTSTL H2-Kd 224, 225 (SEQ. ID NO. 302) Influenza A HA  518-526 IYSTVASSL H2-Kd 224 (SEQ. ID NO. 303) Influenza A HA  259-266 FEANGNLI H2-Kk 226, 227, (SEQ. ID NO.: 304) 228 Influenza A HA   10-18 IEGGWTGMI H2-Kk 226, 227, (SEQ. ID NO.: 305) 228 Influenza A NP   50-57 SDYEGRLI H2-Kk 229, 230 (SEQ. ID NO.. 306) Influenza a NSI  152-160 EEGAIVGEI H2-Kk 231 (SEQ. ID NO.: 307) Influenza NP  336-374 ASNENMETM H2Db  168, 222, A34 (SEQ. ID NO.: 308) 219 Influenza NP  366-374 ASNENMDAM H2Db 232 A68 (SEQ. ID NO.: 309) Influenza B NP   85-94 KLGEFYNQMM HLA-A*0201 233 (SEQ. ID NO.: 310) Influenza B NP   85-94 KAGEFYNQMM HLA-A*0201 234 (SEQ. ID NO.: 311) Influenza HA  204-212 LYQNVGTYV H2Kd 235 JAP (SEQ. ID NO. 312) Influenza HA  210-219 TYVSVGTSTL H2-Kd 225 JAP (SEQ. ID NO.: 313) Influenza HA  523-531 VYQILATYA H2-Kd 235 JAP (SEQ. ID NO. 314) Influenza HA  529-537 IYATVAGSL H2-Kd 235 JAP (SEQ. ID NO. 315) Influenza HA  210-219 TYVSVGTSTI(L > I) H2-Kd 236 JAP (SEQ. ID NO.: 316) Influenza HA  255-262 FESTGNLI H2-Kk 237 JAP (SEQ. ID NO.: 317) JHMV cAg  318-326 APTAGAFFF H2-Ld 238 (SEQ. ID NO.: 318) LCMV NP  118-126 RPQASGVYM H2-Ld 239-240 (SEQ. ID NO. 319) LCMV NP  396-404 FQPQNGQFI H2-Db 241 (SEQ. ID NO. 320) LCMV GP  276-286 SGVENPGGYCL H2-Db 242 (SEQ. ID NO.: 321) LCMV GP   33-42 KAVYNFATCG H2-Db 243, 244 (SEQ. ID NO.: 322) MCMV pp89  168-176 YPHFMPTNL H2-Ld 245 (SEQ. ID NO. 323) MHV spike  510-518 CLSWNGPHL H2-Db 248 protein (SEQ. ID NO. 324) MMTV env gp  474-482 SFAVATTAL H2-Kd 246 36 (SEQ. ID NO.: 325) MMTV gag p27  425-433 SYETFISRL H2-Kd 246 (SEQ. ID NO.: 326) MMTV env gp73  544-551 ANYDFICV H2-Kb 247 (SEQ. ID NO.: 327) MuLV env p15E  574-581 KSPWFTTL H2-Kb 249, 250 (SEQ. ID NO.: 328) MuLV env gp70  189-196 SSWDFITV H2-Kb 251, Sijts (SEQ. ID NO.: 329) et al. Submitted MuLV gag 75K   75-83 CCLCLTVFL H2-Db 252 (SEQ. ID NO.: 330) MuLV env gp70  423-431 SPSYVYHQF H2Ld 253 (SEQ. ID NO.: 331) MV F protein  437-447 SRRYPDAVYLH HLA-B*2705 254 (SEQ. ID NO.: 332) Mv F protein  438-446 RRYPDAVYL HLA-B*2705 255 (SEQ. ID NO. 333) Mv NP  281-289 YPALGLHEF H2-Ld 256 (SEQ. ID NO.: 334) Mv HA  343-351 DPVIDRLYL H2-Ld 257 (SEQ. ID NO. 335) MV HA  544-552 SPGRSFSYF H2-Ld 257 (SEQ. ID NO.: 336) Poliovirus VP1  111-118 TYKDTVQL H2-kd 258 (SEQ. ID NO.: 337) Poliovirus VP1  208-217 FYDGFSKVPL H2-Kd 258 (SEQ. ID NO.: 338) Pseudorabies G111  455-463 IAGIGILAI HLA-A*0201 104 virus gp (SEQ. ID NO.: 339) Rabiesvirus NS  197-205 VEAEIAHQI H2-Kk 227-227 (SEQ. ID NO.: 340) Rotavirus VP7   33-40 IIYRFLLI H2-Kb 259 (SEQ. ID NO.: 341) Rotavirus VP6  376-384 VGPVFPPGM H2-Kb 260 (SEQ. ID NO.: 342) Rotavirus VP3  585-593 YSGYIFRDL H2-Kb 260 (SEQ. ID NO.: 343) RSV M2   82-90 SYIGSINNI H2-Kd 261 (SEQ. ID NO.: 344) SIV  gagp11C  179-190 EGCTPYDTNQML Mamu-A*01 266 (SEQ. ID NO.: 345) SV NP  324-332 FAPGNYPAL H2-Db 262 (SEQ. ID NO.: 346) SV NP  324-332 FAPCTNYPAL H2-Kb 263, 264, (SEQ. ID NO.: 346) 265 SV40 T  404-411 VVYDFLKC H2-Kb 267 (SEQ. ID NO.: 347) SV40 T  206-215 SAINNYAQKL H2-Db 268, 269 (SEQ. ID NO.: 348) SV40 T  223-231 CKGVNKEYL H2-Db 268, 269 (SEQ. ID NO. 349) SV40 T  489-497 QGINNLDNL H2-Db 268, 269 (SEQ. ID NO.: 350) SV40 T  492-500 NNLDNLRDY(L) H2-Db 270 (501) (SEQ. ID NO.: 351) SV40 T  560-568 SEFLLEKRI H2-Kk 271 (SEQ. ID NO. 352) VSV NP   52-59 RGYVYQGL H2-Kb 272 (SEQ. ID NO.: 353)

Table II sets forth antigens identified from various protein sources. The Table is extracted from Table 4.2 in the Rammensee book with the references in Table H being the same as the references in the Rammensee Table 4.2.

TABLE II HLA Class I Motifs HLA-A1 Position (Antigen) Source Ref. T cell epitopes EADPTGHSY MAGE-1 161-169  27, 28 (SEQ. ID NO.: 354) VSDGGPNLY Influenza A PB 1591-599  21, 23 (SEQ. ID NO.: 355) CTELKLSDY Influenza A NP 44-52  23 (SEQ. ID NO.: 356) EVDPIGHLY MAGE-3 168-176  29, 30 (SEQ. ID NO.: 357) HLA-A201 MLLSVPLLLG Calreticulin signal sequence 1-10  34, 35, 36, 37 (SEQ, ID NO.: 358) STBXQSGXQ HBV PRE-S PROTEIN 141-149  43 (SEQ. ID NO.: 359) YMDGTMSQV Tyrosinase 369-377  45 (SEQ, ID NO.: 360) ILKEPVHGV HIV-I RT 476-484   4, 31, 47 (SEQ. ID NO.: 361) LLGFVFTLTV Influenza MP 59-68   4, 39 (SEQ. ID NO,: 362) LLFGYPVYVV HTLV-1 tax 11-19  40 (SEQ. ID NO.: 363) GLSPTVWLSV HBV sAg 348-357  48 (SEQ. ID NO.: 364) WLSLLVPFV HBV sAg 335-343  49, 50, 51 (SEQ. ID NO.: 365) FLPSDFFPSV HBV cAg 18-27  52 (SEQ. ID NO.: 366) C L G 0 L L T M V EBV LMP-2 426-434  48 (SEQ. ID NO.: 367) FLAGNSAYEYV HCMV gp 618-628B  53 (SEQ. ID NO.: 368) KLGEFYNQMM Influenza BNP 85-94  54 (SEQ. ID NO.: 369) KLVALGINAV HCV-1 NS3 400-409  55 (SEQ. ID NO,: 370) DLMGYIPLV HCV MP 17-25  56 (SEQ. ID NO.: 371) RLVTLKDIV HPV 11 EZ 4-12  34, 35 (SEQ. ID NO.: 372) MLLAVLYCL Tyrosinase 1-9  57, 58, 59, 68 (SEQ. ID NO-373) AAGIGILTV Melan A\Mart-127-35  60 (SEQ. ID NO.: 374) YLEPGPVTA Pmel 17/gp 100 480-488  61 (SEQ. ID NO.: 375) ILDGTATLRL Pmel 17/gp 100 457-466  62 (SEQ. ID NO.: 376) LLDGTATLRL Pmel gp1OO 457-466  62 (SEQ. ID NO.: 377) ITDQVPFSV Pmel gp 100 209-217  62 (SEQ. ID NO.: 378) KTWGQYWQV Pmel gp 100 154-162  62 (SEQ. ID NO.: 379) TITDQVPFSV Pmel gp 100 208-217  62 (SEQ. ID NO.: 380) AFHIIVAREL HIV-I nef 190-198  63 (SEQ. ID NO.: 381) YLNKIQNSL P. falciparum CSP 334-342  64 (SEQ. ID NO.: 382) MMRKLAILSV P. falciparum CSP 1-10  64 (SEQ. ID NO.: 383) KAGEFYNQMM Influenza BNP 85-94  65 (SEQ. ID NO.: 384) NIAEGLRAL EBNA-1 480-488  66 (SEQ. ID NO.: 385) NLRRGTALA EBNA-1 519-527  66 (SEQ. ID NO.: 386) ALAIPQCRL EBNA-1 525-533  66 (SEQ. ID NO.: 387) VLKDAIKDL EBNA-1 575-582  66 (SEQ. ID NO.: 388) FMVFLQTHI EBNA-1 562-570  66 (SEQ. ID NO.: 389) HLIVDTDSL EBNA-2 15-23  66 (SEQ. ID NO.: 390) SLGNPSLSV EBNA-2 22-30  66 (SEQ. ID NO. 391) PLASAMRML EBNA-2 126-134  66 (SEQ. ID NO. 392) RMLWMANYI EBNA-2 132-140  66 (SEQ. ID NO.: 393) MLWMANYIV EBNA-2 133-141  66 (SEQ. ID NO.: 394) ILPQGPQTA EBNA-2 151-159  66 (SEQ. ID NO.: 395) PLRPTAPTTI EBNA-2 171-179  66 (SEQ, ID NO.: 396) PLPPATLTV EBNA-2 205-213  66 (SEQ. ID NO.: 397) R M H L P V L H V EBNA-2 246-254  66 (SEQ. ID NO.: 398) PMPLPPSQL EBNA-2 287-295  66 (SEQ. ID NO.: 399) QLPPPAAPA EBNA-2 294-302  66 (SEQ. ID NO.: 400) SMPELSPVL EBNA-2 381-389  66 (SEQ. ID NO.: 401) DLDESWDYI EBNA-2 453-461  66 (SEQ. ID NO.: 402) P L P C V L W P VV BZLF1 43-51  66 (SEQ. ID NO.: 403) SLEECDSEL BZLF1 167-175  66 (SEQ, ID NO.: 404) EIKRYKNRV BZLFI 176-184  66 (SEQ. ID NO.: 405) QLLQFIYREV BZLF1 195-203  66 (SEQ. ID NO.: 406) LLQHYREVA BZLFI 196-204  66 (SEQ. ID NO.: 407) LLKQMCPSL BZLFI 217-225  66 (SEQ. ID NO.: 408) SIIPRTPDV BZLFI 229-237  66 (SEQ. ID NO.: 409) AIMDKNIIL Influenza A NS1 122-130  67 (SEQ. ID NO.: 410) IMDKNIILKA Influenza A NS1 123-132  67 (SEQ. ID NO. 411) LLALLSCLTV HCV MP 63-72  69 (SEQ. ID NO.: 412) ILHTPGCV HCV MP 105-112  69 (SEQ. ID NO.: 413) QLRRHIDLLV HCV env E 66-75  69 (SEQ, ID NO.: 414) DLCGSVFLV HCV env E 88-96  69 (SEQ. ID NO.: 415) SMVGNWAKV HCV env E 172-180  69 (SEQ. ID NO.: 416) HLHQNIVDV HCV NSI 308-316  69 (SEQ. ID NO.: 417) FLLLADARV HCV NSI 340-348  69 (SEQ. ID NO.: 418) GLRDLAVAVEPVV HCV NS2 234-246  69 (SEQ. ID NO.: 419) SLLAPGAKQNV HCV NS1 18-28  69 (SEQ. ID NO.: 420) LLAPGAKQNV HCV NS1 19-28  69 (SEQ. ID NO.: 421) FLLSLGIHL HBV pol 575-583  70 (SEQ. ID NO.: 422) SLYADSPSV HBV pol 816-824  70 (SEQ. ID NO.: 423) GLSRYVARL HBV POL 455-463  70 (SEQ. ID NO.: 424) KIFGSLAFL HER-2 369-377  71 (SEQ. ID NO.: 425) ELVSEFSRM HER-2 971-979  71 (SEQ. ID NO.: 426) KLTPLCVTL HIV-I gp 160 120-128  72 (SEQ. ID NO.: 427) SLLNATDIAV HIV-I GP 160 814-823  72 (SEQ. ID NO.: 428) VLYRYGSFSV Pmel gp100 476-485  62 (SEQ. ID NO.: 429) YIGEVLVSV Non-filament forming class I myosin  73 (SEQ. ID NO.: 430) family (HA-2)** LLFNILGGWV HCV NS4 192-201  74 (SEQ. ID NO.: 431) LLVPFVQWFW HBV env 338-347  74 (SEQ. ID NO.: 432) ALMPLYACI HBV pol 642-650  74 (SEQ. ID NO.: 433) YLVAYQATV HCV NS3 579-587  74 (SEQ. ID NO.: 434) TLGIVCPIC HIPV 16 E7 86-94  74 (SEQ. ID NO.: 435) YLLPRRGPRL HCV core protein 34-43  74 (SEQ. ID NO.: 436) LLPIFFCLWV HBV env 378-387  74 (SEQ. ID NO.: 437) YMDDVVLGA HBV Pol 538-546  74 (SEQ. ID NO.: 438) GTLGIVCPI HPV16 E7 85-93  74 (SEQ. ID NO.: 439) LLALLSCLTI HCV MP 63-72  74 (SEQ. ID NO.: 440) MLDLQPETT HPV 16 E7 12-20  74 (SEQ. ID NO.: 441) SLMAFTAAV HCV NS4 174-182  75 (SEQ. ID NO.: 442) CINGVCWTV HCV NS3 67-75  75 (SEQ. ID NO.: 443) VMNILLQYVV Glutarnic acid decarboxylase  76 (SEQ. ID NO.: 444) 114-123 ILTVILGVL Melan A/Mart- 32-40  77 (SEQ. ID NO.: 445) FLWGPRALV MAGE-3 271-279  78 (SEQ. ID NO.: 446) L L C P A G H A V HCV NS3 163-171  54 (SEQ. ID NO.: 447) ILDSFDPLV HCV NSS 239-247  54 (SEQ. ID NO.: 448) LLLCLIFLL HBV env 250-258  79 (SEQ. ID NO.: 449) LIDYQGMLPV HBV env 260-269  79 (SEQ. ID NO.: 450) SIVSPFIPLL HBV env 370-379  79 (SEQ. ID NO.: 451) FLLTRILTI HBV env 183-191  80 (SEQ, ID NO.: 452) HLGNVKYLV P. faciparum TRAP 3-11  81 (SEQ, ID NO.: 453) GIAGGLALL P. faciparum TRAP 500-508  81 (SEQ. ID NO.: 454) ILAGYGAGV HCV NS S4A 236-244  82 (SEQ. ID NO.: 455) GLQDCTMLV HCV NS5 714-722  82 (SEQ. ID NO.: 456) TGAPVTYSTY HCV NS3 281-290  83 (SEQ. ID NO.: 457) VIYQYMDDLV HIV-1RT 179-187  84 (SEQ, ID NO.: 458) VLPDVFIRCV N-acetylglucosaminyltransferase V  85 (SEQ, ID NO.: 459) Gnt-V intron VLPDVFIRC N-acetylglucosaminyltransferase V  85 (SEQ. ID NO.: 460) Gnt-V intron AVGIGIAVV Human CD9  86 (SEQ. ID NO.: 461) LVVLGLLAV Human glutamyltransferase  86 (SEQ, ID NO.: 462) ALGLGLLPV Human G protein coupled receptor  86 (SEQ, ID NO.: 463) 164-172 GIGIGVLAA HSV-1 gp C 480-488  86 (SEQ. ID NO.: 281) GAGIGVAVL HSV-2 gp C 446-454  86 (SEQ. ID NO.: 464) IAGIGILAI Pseudorabies gpGIN 455-463  86 (SEQ. ID NO.: 465) LIVIGILIL Adenovirus 3 E3 9 kD 30-38  86 (SEQ. ID NO.: 466) LAGIGLIAA S. Lincolnensis ImrA  86 (SEQ. ID NO.: 467) VDGIGILTI Yeast ysa-1 77-85  86 (SEQ. ID NO.: 468) GAGIGVLTA B. polymyxa, βcndoxylanase 149-  86 (SEQ. ID NO.: 469) 157 AAGIGIIQI E. coli methionine synthase 590-598  86 (SEQ. ID NO.: 470) QAGIGILLA E. coli hypothetical protein 4-12  86 (SEQ. ID NO.: 471) KARDPHSGHFV CDK4w1 22.32  87 (SEQ. ID NO.: 472) KACDPI-ISGIIFV CDK4-R24C 22-32  87 (SEQ. ID NO.: 473) ACDPFISGHFV CDK4-R24C 23-32  87 (SEQ. ID NO.: 474) SLYNTVATL HIV-I gag p 17 77-85  99 (SEQ. ID NO.: 475) ELVSEFSRV HER-2, m > V substituted 971-979  89 (SEQ. ID NO.: 476) RGPGRAFVTI HIV-I gp 160 315-329  90 (SEQ. ID NO.: 477) HMWNFISGI HCV NS4A 149-157  91 (SEQ. ID NO.: 478) NLVPMVATVQ HCMV pp65 495-504  92 (SEQ. ID NO.: 479) GLHCYEQLV HPV 6b E7 21-30  93 (SEQ. ID NO.: 480) PLKQHFQIV HPV 6b E7 47-55  93 (SEQ. ID NO.: 481) LLDFVRFMGV EBNA-6 284-293  95 (SEQ. ID NO.: 482) AIMEKNIML Influenza Alaska NS 1 122-130  67 (SEQ. ID NO.: 483) YLKTIQNSL P. falciparum cp36 CSP  96 (SEQ. ID NO.: 484) YLNKIQNSL P. falciparum cp39 CSP  96 (SEQ. ID NO.: 485) YMLDLQPETT HPV 16 E7 11-20*  97 (SEQ, ID NO.: 486) LLMGTLGIV HPV16 E7 82-90**  97 (SEQ. ID NO.: 487) TLGIVCPI HPV 16 E7 86-93  97 (SEQ. ID NO.: 488) TLTSCNTSV HIV-1 gp120 197-205  98 (SEQ. ID NO.: 489) KLPQLCTEL HPV 16 E6 18-26  97 (SEQ. ID NO.: 490) TIHDIILEC HPV16 E6 29-37  97 (SEQ. ID NO.: 491) LGIVCPICS HPV16 E7 87-95  97 (SEQ. ID NO.: 492) VILGVLLLI Melan A/Mart-1 35-43  68 (SEQ. ID NO.: 493) ALMDKSLHV Melan A/Mart-1 56-64  68 (SEQ. ID NO.: 494) GILTVILGV Melan A/Mart-1 31-39  68 (SEQ. ID NO.: 495) T cell epitopes MINAYLDKL P. Falciparum STARP 523-531  81 (SEQ. ID NO.: 496) AAGIGILTV Melan A/Mart- 127-35 100 (SEQ. ID NO.: 497) FLPSDFFPSV HBV cAg 18-27  51 (SEQ. ID NO,: 498) Motif unknown SVRDRLARL EBNA-3 464-472 101 T cell epitopes (SEQ. ID NO.: 499) T cell epitopes AAGIGILTV Melan A/Mart-1 27-35 100 (SEQ. ID NO.: 497) FAYDGKDYI Human MHC 1-ot 140-148  99 (SEQ. ID NO.: 500) T cell epitopes AAGIGILTV Melan A/Mart-1 27-35 100 (SEQ, ID NO.: 497) FLPSDFFPSV HBV cAg 18-27  51 (SEQ. ID NO.: 498) Motif unknown AAGIGILTV Meland A/Mart-1 27-35 100 T cell epitopes (SEQ. ID NO.: 497) FLPSDFFPSV HBV cAg 18-27  51 (SEQ. ID NO.: 498) AAGIGILTV Melan A/Mart-1 27-35 100 (SEQ. ID NO.: 497) ALLAVGATK Pmel17 gp 100 17-25 107 (SEQ. ID NO.: 501) T cell epitopes R L R D L L L I V T R HIV-1 gp41 768-778 108 (SEQ. ID NO.: 502) QVPLRPMTYK HIV-1 nef 73-82 109 (SEQ. ID NO.: 503) TVYYGVPVWK HIV-1 gp120-36-45 110 (SEQ. ID NO.: 504) RLRPGGKKK HIV-1 gag p 17 20-29 110 (SEQ. ID NO.: 505) ILRGSVAHK Influenza NP 265-273  21 (SEQ. ID NO,: 506) RLRAEAGVK EBNA-3 603-611 111 (SEQ. ID NO.: 507) RLRDLLLIVTR HIV-1 gp41 770-780 112 (SEQ. ID NO.: 502) VYYGVPVWK HIV-I GP 120 38-46 113 (SEQ. ID NO.: 508) RVCEKMALY HCV NS5 575-583 114 (SEQ. ID NO.: 509) Motif unknown KIFSEVTLK Unknown; muta melanoma peptide Wolfel et al., T cell epitope (SEQ, ID NO.: 510) ted (p 1 83L) 175-183 pers. Comm. YVNVNMGLK* HBV cAg 88-96 116 (SEQ. ID NO.: 511) T cell epitopes IVTDFSVIK EBNA-4 416-424 115, 117 (SEQ. ID NO.: 512) ELNEALELK P53 343-351 115 (SEQ, ID NO.: 513) VPLRPMTYK HIV-1 NEF 74-82 115 (SEQ. ID NO.: 514) AIFQSSMTK HIV-1 gag p24 325-333 115 (SEQ. ID NO.: 515) QVPLRPMTYK HIV-1 nef 73-82 118 (SEQ. ID NO.: 516) TINYTIFK HCV NSI 238-246 114 (SEQ. ID NO.: 517) AAVDLSHFLKEK HIV-1 nef 83-94 120 (SEQ. ID NO.: 518) ACQ G V G G P G G H K HIV-1 I I 1B p24 349-359 122 (SEQ, ID NO,: 519) HLA-A24 S Y L D S G I H F* β-catenin, mutated 123 (SEQ, ID NO.: 520) (proto-onocogen) 29-37 T cell epitopes RYLKDQQLL HIV GP 41 583-591 124 (SEQ. ID NO.: 521) AYGLDFYIL P15 melanoma Ag 10-18 125 (SEQ. ID NO.: 522) AFLPWHRLFL Tyrosinase 206-215 126 (SEQ. ID NO.: 523) AFLPWHRLF Tyrosinase 206-214 126 (SEQ. ID NO.: 524) RYSIFFDY Ebna-3 246-253 101 (SEQ. ID NO.: 525) T cell epitope ETINEEAAEW HIV-1 gag p24 203-212 127 (SEQ. ID NO.: 526) T cell epitopes STLPETTVVRR HBV cAg 141-151 129 (SEQ. ID NO,: 527) MSLQRQFLR ORF 3P-gp75 294-321 (bp) 130 (SEQ, ID NO.: 528) LLPGGRPYR TRP (tyrosinase rel.) 197-205 131 (SEQ. ID NO.: 529) T cell epitope IVGLNKIVR HIV gag p24 267-267-275 132, 133 (SEQ. ID NO.: 530) AAGIGILTV Melan A/Mart- 127 35 100 (SEQ. ID NO.: 531)

Table III sets forth additional antigens useful in the invention that are available from the Ludwig Cancer Institute. The Table refers to patents in which the identified antigens can be found and as such are incorporated herein by reference. TRA refers to the tumor-related antigen and the LUD No. refers to the Ludwig Institute number.

TABLE III LUD Date Patent TRA No. Patent No. Issued Peptide (Antigen) HLA MAGE-4 5293 5,405,940 11 Apr. 1995 EVDPASNTY HLA-A1 (SEQ. ID NO.: 532) MAGE-41 5293 5,405,940 11 Apr. 1995 EVDPTSNTY HLA-A1 (SEQ ID NO: 533) MAGE-5 5293 5,405,940 11 Apr. 1995 EADPTSNTY HLA-A1 (SEQ ID NO: 534) MAGE-51 5293 5,405,940 11 Apr. 1995 EADPTSNTY HLA-A1 (SEQ ID NO: 534) MAGE-6 5294 5,405,940 11 Apr. 1995 EVDPIGHVY HLA-A1 (SEQ ID NO: 535) 5299.2 5,487,974 30 Jan. 1996 MLLAVLYCLL HLA-A2 (SEQ ID NO: 536) 5360 5,530,096 25 Jun. 1996 MLLAVLYCL HLA-B44 (SEQ ID NO: 537) Tyrosinase 5360.1 5,519,117 21 May 1996 SEIWRDIDFA HLA-B44 (SEQ ID NO: 538) SEIWRDIDF (SEQ ID NO: 539) Tyrosinase 5431 5,774,316 28 Apr. 1998 XEIWRDIDF HLA-B44 (SEQ ID NO: 540) MAGE-2 5340 5,554,724 10 Sep. 1996 STLVEVTLGEV HLA-A2 (SEQ ID NO: 541) LVEVTLGEV (SEQ ID NO: 542) VIFSKASEYL (SEQ ID NO: 543) IIVLAIIAI (SEQ ID NO: 544) KIWEELSMLEV (SEQ ID NO: 545) LIETSYVKV (SEQ ID NO: 546) 5327 5,585,461 17 Dec. 1996 FLWGPRALV HLA-A2 (SEQ ID NO: 547) TLVEVTLGEV (SEQ ID NO: 548) ALVETSYVKV (SEQ ID NO: 549) MAGE-3 5344 5,554,506 10 Sep. 1996 KIWEELSVL HLA-A2 (SEQ ID NO: 550) MAGE-3 5393 5,405,940 11 Apr. 1995 EVDPIGHLY HLA-A1 (SEQ ID NO: 551) MAGE 5293 5,405,940 11 Apr. 1995 EXDX5Y HLA-A1 (SEQ. ID NO.: 552) (but not EADPTGHSY) (SEQ. ID NO.: 553) E(A/V)D X5 Y (SEQ. ID NO.: 554) E(A/V)D P X4 Y (SEQ. ID NO.: 555) E(A/V)D P(I/A/T)X3 Y (SEQ. ID NO.: 556) E(A/V)D P(I/A/T)(G/S)X2 Y (SEQ. ID NO.: 557) E(A/V)D P(I/A/T)(G/S)(H/N)X Y E (A/V) DP (I/A/T) (G/S) (H/N) (L/T/V)Y (SEQ. ID NO.: 559) MAGE-1 5361 5,558,995 24 Sep. 1996 ELHSAYGEPRKLLTQD HLA-C (SEQ ID NO: 560) Clone 10 EHSAYGEPRKLL (SEQ ID NO: 561) SAYGEPRKL (SEQ ID NO: 562) MAGE-1 5253.4 TBA TBA EADPTGHSY HLA-A1 (SEQ ID NO: 563) BAGE 5310.1 TBA TBA MAARAVFLALSAQLLQARLMKE HLA-C (SEQ ID NO: 564) Clone 10 MAARAVFLALSAQLLQ HLA-C (SEQ ID NO: 565) Clone 10 AARAVFLAL HLA-C (SEQ ID NO: 566) Clone 10 GAGE 5323.2 5,648,226 15 Jul. 1997 YRPRPRRY HLA-CW6 (SEQ. ID NO.: 567)

Preferred peptide antigens are those of tumor associated antigens (TAA) and chronic infections. Particularly preferred peptide antigens are derived from tyrosinose, gp100 or Melan A for the treatment of melanoma.

The peptide antigens of this invention are readily prepared using standard peptide synthesis means known in the art. Generally they can be prepared commercially by one of numerous companies that do chemical synthesis. An example is American Peptides, Inc., where the distributor is CLINALFA AG (Laufelfingen, Switzerland). The antigens are prepared in accordance with GMP standards. Purity is assessed by analytical HPLC. The product is characterized by amino-acid analysis and tested for sterility and the absence of pyrogens.

In delivering an appropriate antigen, e.g., a polypeptide, to the animal's system it may be delivered directly as the polypeptide, or it may be delivered indirectly, e.g., using a DNA construct or vector, or a recombinant virus that codes for the desired antigen. Any vector driving expression in a professional antigen presenting cell is suitable for this purpose. In the indirect delivery, the antigen is expressed in the cell, to be presented by the MHC Class I on the surface of the cell to stimulate the CTL response.

In a preferred embodiment of the invention an encoded antigen can be delivered in the form of a naked plasmid expression vector. Particularly useful constructs are disclosed in U.S. patent application Ser. No. 09/561,572 entitled EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS which is incorporated herein by reference in its entirety. The feasibility of and general procedures related to the use of naked DNA for immunization are described in U.S. Pat. Nos. 5,589,466, entitled “INDUCTION OF A PROTECTIVE IMMUNE RESPONSE IN A MAMMAL BY INJECTING A DNA SEQUENCE” and 5,679,647, entitled “METHODS AND DEVICES FOR IMMUNIZING A HOST AGAINST TUMOR-ASSOCIATED ANTIGENS THROUGH ADMINISTRATIONS OF NAKED POLYNUCLEOTIDES WHICH ENCODE TUMOR-ASSOCIATED ANTIGENIC PEPTIDES” which are herein incorporated by reference in their entirety. However the former teaches only intramuscular or intradermal injection while the latter teaches only administration to skin or mucosa. Administration directly to the lymphatic system is greatly more efficient (see examples 6-9, below). Single bolus injection into lymph node required only 0.1% of the dose required in order to obtain a similar level of CTL response by intramuscular (i.m.) injection. It is therefore feasible to establish a protective response against systemic viral infection with a single bolus delivered i.ln., but not with a dose nearing the practical limit delivered i.m. Even repeated bolus injections i.m. failed to establish a protective response against a peripheral virus infection or transplanted tumor whereas lower doses administered i.m. were completely effective.

In another embodiment of the invention an encoded antigen can be delivered in the form of a viral vector. A wide array of viruses with modified genomes adapted to express interposed reading frames but often no, or at least a reduced number of, viral proteins are known in the art, including without limitation, retroviruses including lentiviruses, adenoviruses, parvoviruses including adeno-associated virus, herpesviruses, and poxviruses including vaccinia virus. Such viral vectors facilitate delivery of the nucleic acid component into the cell allowing for expression. A subset of these vectors, such as retroviruses and parvoviruses, also promotes integration of their nucleic acid component into the host genome, whereas others do not.

Bacteria can also serve as vectors, analogously to viruses, i.e. they can be used to deliver a nucleic acid molecule capable of causing expression of an antigen. For example, a strain of Listeria monocytogenes has been devised that effects its own lysis upon entering the cytosol of macrophages (its normal target), thereby releasing plasmid from which antigen was subsequently expressed (Dietrich, G. et al. Biotechnology 16:181-185, 1998 which is herein incorporated by reference in their entirety). Shigela flexneri and Escherichia coli have been similarly used (Sizemore, D. R. et al. Science 270:299-302, 1995, and Courvalin, P. et al. Life Sci. 318:1207-1212, 1995, respectively, which are herein incorporated by reference in their entirety).

The use of microbial vectors for nucleic acid delivery can be complicated by the immune reactions the vectors themselves provoke. When prolonged or repeated administration is required, antibody elicited by the earlier treatment can prevent useful quantities of the vector from ever reaching its intended host. However, by direct administration into, for example, a lymph node, the combination of proximity to host cells and the much reduced effective dose makes it possible to administer a dose capable of evading or overwhelming an existing antibody titer.

The word vector has been used, here and elsewhere, in reference to several modalities and variously modified (e.g. expression vector, viral vector, delivery vector, etc.). The underlying principle here is that a nucleic acid capable of causing expression of an antigen ultimately arrives in an APC, rather than the antigen itself. Unless modified, explicitly or by local context, wherever the term vector is used herein, it is intended to encompass all such possibilities.

These foregoing techniques are distinct from the approach of modifying the microbial genome (including extra-chromosomal DNA) so that the antigen is produced as a component of the microbe (virus, bacteria, fungi, protazoan, etc., etc.), which is then itself administered as the immunogen. Obviously, the genomic modification would most likely involve the use some reagent falling within the above definition of vector. Again, the distinction is whether the vaccine includes the already synthesized antigen, or a nucleic acid capable of causing an APC to express the antigen in vivo. This strategy constitutes a further embodiment of the invention. For rhetorical clarity we have discussed these approaches as if they were mutually exclusive, but in fact combinations are possible, e.g., a virus vector as above that also incorporates a target epitope into a capsid or envelope protein.

Similarly, antigen presenting cells can also be manipulated in vitro and then themselves used as the active component of a vaccine. Antigen expression can be conferred by delivering nucleic acid encoded antigen using any of the transduction techniques known in the art, including without limitation electroporation, viral or bacterial transduction, lipid-mediated transduction, and biolistic bombardment. Alternatively the APCs may simply be pulsed with antigen. As with any of the other embodiments of this invention an antigen can be an approximately 8-10 amino acid peptide representing a single epitope, a complete protein, a polypeptide encompassing one or more epitopes (including epitopes originally derived from multiple proteins) or other forms of antigen described above.

Antigens may be used alone or may be delivered in combination with other antigens or with other compounds such as cytokines that are known to enhance immune stimulation of CTL responses, such as, GM-CSF, IL-12, IL-2, TNF, IFN, IL-18, IL-3, IL-4, IL-8, IL-9, IL-13, IL-10, IL-14, IL-15, G-SCF, IFN alpha, IFN beta, IFN gamma, TGF alpha, TGF beta, and the like. The cytokines are known in the art and are readily available in the literature or commercially. Many animal and human tumors have been shown to produce cytokines such as IL-4, IL-10, TGF-B that are potent modulators of the immune response and that protect tumors from immune-mediated destruction. The production of IL-4, IL-10 or TGF-B by the tumors may achieve this protective effect by suppressing the induction of cellular immunity, including the elaboration of CTL responses. Alternatively, cytokines that support CTL responses can be exogenously added to help in the balance between induction of anti-tumor cell mediated and non-tumor-destructive humoral responses. Several such exogenous cytokines show utility in experimental mouse vaccination models which are known to enhance CTL responses, including GM-CSF, IFN and IL-2. An effective exogenous cytokine that may be used is GM-CSF. GM-CSF is reported to enhance the expression of the so called “co-stimulatory” molecules, such as B7-1 or B7-2 on antigen presenting cells (APC), which are important players in the symphony of interactions that occur during stimulation of CTL by APC. Moreover, GM-CSF is known to induce activation of APC and to facilitate growth and differentiation of APC, thereby making these important CTL stimulating cells available both in greater numbers and potency.

Delivery of the Antigen

This invention is based in part on the observation that a CTL response is not sustained using standard vaccine techniques. While not wanting to be bound by any particular theory, it is thought that T cells do not have a functional memory that is long-lived. Antibody-mediated B-cell memory, on the other hand, appears to have a long-lived effector memory. Thus, delivering an antigen that produces a CTL response must be done over time to keep the patient's immune system appropriately stimulated to attack the target cells. While it has been suggested that antigens and adjuvants can be prepared as biodegradable microspheres or liposomes, none of these preparations have thus far provided a CTL response that is useful for attacking cancer cells or pathogens on a long term basis. The delivery must be sustained over the desired period of time at a level sufficient to maintain the antigen level to obtain the desired response and that it must be delivered from a reservoir having fluid antigen composition that is introduced so that it reaches the animal's lymphatic system.

Ultimately antigen must find its way into the lymphatic system in order to efficiently stimulate CTL. However, delivery of antigen according to the invention can involve infusion into various compartments of the body, including but not limited to subcutaneous, intravenous, intraperitoneal and intralymphatic, the latter being preferred. Each of these various points of infusion results in antigen uptake into the lymphatic system. The relative amounts of antigen needed to induce a beneficial CTL response varies according to the different sites of infusion. In general, direct infusion of antigen into the lymph system is deemed to be the most efficient means of inducing a CTL response, but that the material difference between the various routes is not necessarily relevant in terms of the quantity of antigen needed, or, in terms of the operating parameters of the invention. The pump systems of the invention are capable of delivering material quantities of antigen in a range that makes the invention suitable for inducing CTL response through delivery to all compartments of the body. CTL stimulation based on delivery of antigen via various routes will be variable, based on the properties of different antigens, which will reflect factors that influence antigen behavior in the body and its rate of equilibration to (or longevity in) the lymph, such an antigen stability in the body fluid, solubility of antigen in body fluid, binding affinity for HLA and potency as a stimulator of CTL.

It is most efficient, and therefore, preferred, that the introduction is done as directly as possible to the lymphatic system to avoid the destruction of the antigen by metabolism in the body. When introduction of a fluid antigen composition occurs subcutaneously, larger quantities of antigen are needed to assure enough antigen reaches the lymphatic system. Such subcutaneous injection is contemplated by this invention if it can be justified by factors such as cost, stability of the antigen, how quickly the antigen gets to the lymph system, how well it equilibrates with the lymph, and other factors that the attending doctor or specialist will recognize. Subcutaneous delivery will generally require 100 to 1000 times more antigen than direct delivery to the lymph system. It is preferable, therefore, that the antigen composition is introduced through a device for local administration to the lymphatic system, e.g. the spleen, a lymph node, or a lymph vessel. The device for local administration may be positioned outside the patient or implanted into the patient. In either case, the device will have a reservoir to hold the fluid antigen-containing composition, a pump to transfer the composition, and a transmission channel leading from the reservoir to be directed to the preferred region of administration in the patient's body. In either case it is preferably portable.

For the device positioned outside the patient's body (the external device), there are numerous devices used for delivering insulin to diabetic patients that are useful in this invention. Generally these are comprised of a reservoir for holding the antigen composition (instead of insulin), a programmable pump to pump the composition out of the reservoir, a transmission channel or line for transmitting the composition, and a means to introduce the composition into the animal's body to ultimately reach the lymphatic system.

The pump employed may be a roller/peristaltic pump, a syringe pump, a piston/valve pump, a gas pressure pump, or the like that has a power source (generally a battery for portability) that is programmable to deliver the desired level of antigen composition to the patient's body and the lymphatic system. A further discussion of the operation of these pumps may be found “Insulin Pump Therapy” by E. Austenst and T. Stahl, Walter de Gruyter, Berlin, N.Y. (1990), at Chapter 3. A list of pumps available at that time that are useful for this invention are given in Table IV.

More recent versions of these pumps are available from the manufacturers shown.

TABLE IV Name Manufacturer/distributor Weight (g) Size (mm) Nordisk Infusor Nordisk 180 100 × 60 × 20  Betatron I CPI/Lilly 197 99 × 66 × 20 RW 90 P/RW 91 Dahedi/EA Satorius 110 109 × 42 × 22  P/RW 92 Instruments MRS 4-Infuser Disetronic 100 75 × 53 × 19 B-D 1000 Becton-Dickinson 131 7  8 × 57 × 20 Nordisk Infusor Nordisk 180 113 × 65 × 22  MK 11 MRS 3-Infuser Disetronic 100 75 × 53 × 18 A S8 MP Autosyringe/Travenol 161 102 × 64 × 19  Betatron 11 CPULilly 197 99 × 66 × 20 Minimed 504 Pacesetter/Haselmeyer 106 86 × 21 × 51 Minimed 404 S* Pacesetter 106 86 × 21 × 51 MRS I/H-Tron Disetronic/Hoechst 100 75 × 53 × 18 *not yet commercially available

Particularly useful pumps are the Disetronic H-Tron V 100 Insulin Pump from Disetronic Medical Systems, Burgdorf, Switzerland and the Minimed 507 Insulin Pump from MiniMed Inc., 12744 San Fernando Road, Sylmar, Calif. 91342. The MiniMed is particularly useful in that it allows programming a bolus without looking at the pump through a series of audio tones (settable in either 0.5 or 1.0 unit increments) and allows programming a bolus for delivery over an extended period of time—from 30 minutes to 4 hours. It provides up to 12 basal rates (or profiles) that can be programmed per 24 hours from 0.0-25 units/hour in 0.1 unit increments. The device allows for the temporary increase or decrease of a set basal rate from 30 minutes to 24 hours in 30 minute increments, Other features relating to safety, time display, memory, etc. are available from the manufacturer.

The reservoir for the antigen composition should be large enough for delivery of the desired amount of antigen over time and is easily refillable or replaceable without requiring the user to reinsert the means for introducing the antigen composition to the lymph system.

In preparing the antigen compositions of this invention, a composition (preferably aqueous) is prepared to be compatible with the lymph system and is physiologically acceptable to the animal being treated. In preparing the antigen compositions useful in this invention one considers the physicochemical properties of the antigen such as the isoelectric point, molecular weight, glycosylation or other post-translational modification, and overall amino acid composition. These properties along with any known behavior of the drug in different solutions (e.g. different buffers, cofactors, etc.) as well as its in vivo behavior will help guide the choice of formulation components. One parameter that impacts all the major degradation pathways is the solution pH. Thus, the initial formulations also assess the pH dependence of the degradation reactions and the mechanism for degradation can often be determined from the pH dependence to determine the stability of the protein in each solution. Rapid screening methods usually involve the use of accelerated stability at elevated temperatures (e.g. 40° C.) using techniques known in the art.

In general the antigen compositions useful in this invention will be prepared suitable for parenteral injection, in very small quantities. As such a composition must be free of contamination and have a pH compatible with the lymph system. However, because very small quantities of the antigenic composition will be delivered it need not be the same pH as blood or lymph, and it need not be aqueous-based. For antigens that are less soluble a suitable cosolvent or surfactant may be used, such as dimethyl sulfoxide (DMSO) or PLURONIC brand surfactants. The pH range that is compatible is from about 6.7-7.3 and can be prepared using water for injection to meet USP specifications (see Remington: The Science and Practice of Pharmacy, Nineteenth Edition; Chapters 86-88). Generally, a standard saline solution that is buffered with a physiologically acceptable weak acid and its base conjugate, e.g., a phosphate or citrate buffering system, will be the basis of the antigen composition. In some cases, a small amount of an antioxidant may be useful to stabilize the composition and prevent oxidation. Factors to consider in preparing the antigen compositions may be found in the 1994 American Chemical Society book entitled “Formulation and Delivery of Proteins and Peptides” (Acs Symposium Series, No. 567) by Jeffery L. Cleland and Robert Langer (Editor)).

For nucleic acid encoded antigens similar considerations apply, although the variety of physico-chemical properties encountered with polypeptides is absent, so that acceptable formulations will have nearly universal applicability. As seen in examples 6-10, plasmid DNA in standard phosphate buffered saline (PBS) is an acceptable and effective formulation. In some embodiments of the invention, DNA is administered continuously or intermittently at short intervals, from a reservoir worn on, or implanted in, the patient's body. It is preferable that the DNA be maintained in a soluble, stable form at or near body temperature over a period of time measured minimally in days. In such applications where the formulated nucleic acid will be delivered from a reservoir over a period several days or longer, the stability of the nucleic acid at room or body temperature for that period of time, as well as its continued sterility, take on increased importance. The addition of bacteriostatic agents (e.g. benzyl or ethyl alcohol) and chelating agents (e.g. EDTA) is useful toward these ends. Formulations containing about 1-10% ethyl alcohol, 0-1% benzyl alcohol, 0.25-0.5 mM EDTA and a citrate-phosphate buffer of pH 7.4-7.8 generally perform well. Such formulations are also appropriate for bolus injections.

Generally the amount of the antigen in the antigen composition will vary from patient to patient and from antigen to antigen, depending on such factors as the activity of the antigen in inducing a response and the flow rate of the lymph through the patient's system. In general the antigen composition may be delivered at a rate of from about 1 to about 500 microliters/hour or about 24 to about 12000 microliters/day. The concentration of the antigen is such that about 0.1 micrograms to about 10,000 micrograms of the antigen will be delivered during 24 hours. The flow rate is based on the knowledge that each minute approximately about 100 to about 1000 microliters of lymph fluid flows through an adult inguinal lymph node. The objective is to maximize local concentration of vaccine formulation in the lymph system. A certain amount of empirical investigation on patients will be necessary to determine the most efficacious level of infusion for a given vaccine preparation in humans.

To introduce the antigen composition into the lymphatic system of the patient the composition is preferably directed to a lymph vessel, lymph node, the spleen, or other appropriate portion of the lymph system. Preferably, the composition is directed to a lymph node such as an inguinal or axillary node by inserting a catheter or needle to the node and maintaining the catheter or needle throughout the delivery. Suitable needles or catheters are available made of metal or plastic (e.g. polyurethane, polyvinyl chloride [PVC], TEFLON, polyethylene, and the like). In inserting the catheter or needle into the inguinal node for example, the inguinal node is punctured under ultrasonographic control using a Vialon™ Insyte-W™ cannula and catheter of 24G3/4 (Becton Dickinson, USA) which is fixed using Tegadenn transparent dressing Tegaderm™ 1624, 3M, St. Paul, Minn. 55144, USA). This procedure is generally done by an experienced radiologist. The location of the catheter tip inside the inguinal lymph node is confirmed by injection of a minimal volume of saline, which immediately and visibly increases the size of the lymph node. The latter procedure allows confirmation that the tip is inside the node and can be performed to ensure that the tip does not slip out of the lymph node can be repeated on various days after implantation of the catheter. In case the tip did in fact slip out of location inside the lymph node, a new catheter can be implanted.

In another embodiment, the antigen is delivered to the lymphatic system through an article of manufacture that is implanted in the animal, preferably at or near a site of a lymphatic organ. The article will include a pump that can deliver the antigen at a controlled rate over a pre-determined period of time and is suitable for use in the host. Several devices are known in the art for the delivery of agents (such as drugs) in humans or animals and these can be used or adapted for use in the present invention.

The implantable device will be similar to the external device discussed above in that it comprises a reservoir of a physiologically-acceptable, aqueous, antigen-containing composition that is capable of inducing a CTL response in an animal, a pump positioned in association with the reservoir to deliver the composition at a defined rate, a transmission channel to discharge the composition from the reservoir, and optionally a delivery line connected to the transmission channel, which delivery line is of a size suitable for positioning in the animal and for delivery of the composition in a manner that reaches the lymphatic system of the animal.

Preferably the pump in the implantable device is an osmotic pump of the type used in the ALZET® model device or the DUROS™ model device pioneered by Alza Corporation, Palo Alto, Calif. or in a device made by Pharmetrix and exemplified in U.S. Pat. No. 4,838,862. The osmotic pump utilizes the osmotic effect using a membrane permeable to water but impermeable to a solute. Osmotic pressure built up in a device is used to deliver a composition at a controlled rate over time. A review by Giancarlo Santus and Richard Baker of “Osmotic Drug Delivery: A Review of the Patent Literature” in the Journal of Controlled Release 35 (1995) 1-21, provides useful guidelines for the type of osmotic pumps that are useful in this invention. The osmotic pump forces the composition through a discharge orifice to discharge the composition, Optionally a delivery line connects to the discharge orifice to position the line suitably for delivery to the lymphatic system of the animal. Patents that describe devices useful in this invention include the following U.S. Pat. Nos. (A) 3,604,417 assigned to American Cyanamid; (B) 4,838,862; 4,898,582; 5,135,498; 5,169,390; and 5,257,987 all assigned to Pharmetrix, (C) 4,340,048; 4,474,575; 4,552,651; 4,619,652; 4,753,651; 3,732,865; 3,760,804; 3,760,805; 3,929,132; 3,995,632; 4,034,756; 4,350,271; 4,455,145; 5,017,381; 5,023,088; 5,030,216; 5,034,229; 5,037,420; 5,057,318; 5,059,423; 5,110,596; 5,110,597; 5,135,523; 5,137,727; 5,174,999; 5,209,746; 5,221,278; 5,223,265; 3,760,984; 3,987,790; 3,995,631; 4,203,440; 4,286,067; 4,300,558; 4,304,232; 4,340,054; 4,367,741; 4,450,198; 4,855,141; 4,865,598; 4,865,845; 4,872,873; 4,929,233; 4,963,141; 4,976,966, all assigned to Alza Corp. Each of the foregoing patents are incorporated herein by reference.

A basic osmotic pump device incorporates a housing containing a chamber for storing the antigen containing composition to be delivered, separated from a compartment containing an osmotic salt material by a barrier that is moveable under pressure such as a piston or a flexible impermeable membrane. The compartment containing the osmotic salt is separated from osmotic fluid by a semipermeable membrane. In some embodiments, a fluid barrier, such as a foil sheet, isolates the osmotic salt chamber from the osmotic fluid, keeping the pump inactivated until removal of the barrier immediately prior to use. Other osmotic pump devices use body fluid as the osmotic fluid. In these devices a semipermeable membrane separates the osmotic salt compartment from body fluids, and the pump is activated once inserted into the body under exposure to body fluids. In either case, volumetric expansion of the osmotic salt compartment drives the expulsion of the stored antigen from the compartment and into the surrounding environment of the body. These pumps have been highly successful at achieving steady-state pumping and delivery of agents. The pumps are of a small size that can be inserted into a patient, with flexible consideration as to location. This is important in the case of CTL vaccines, since the inventor has determined that efficient induction of CTL responses is contingent on the antigen or antigen expression system being delivered into the lymphatic system, in order to ultimately achieve antigen delivery into a lymphatic organ such as the spleen. Antigen delivered into a lymph node is 100-1000 times more efficient at inducing CTL responses compared with conventional subcutaneous delivery. A modification to the osmotic pump incorporates a microcatheter attachment (i.e., the optional delivery line) at its discharge end, such that when the pump is implanted proximal to a lymphatic organ, such as a lymph node, the catheter can be inserted into the organ to facilitate delivery of the vaccine directly into the lymphatic system.

Prior to the administration of the antigen using any of the above vehicles, methods may be used to assist in the determination of the optimum location for the antigen delivery. For example, when using the osmotic pump, radiography may be used to image a patient's lymphatic flow, to determine where relatively high lymphatic drainage occurs, in order to decide upon an insertion position for the osmotic pump that maximizes delivery into the lymphatic system. Since each patient has unique lymphatic drainage profiles, imaging would be conducted for each individual prior to insertion of osmotic pump for delivery of antigen. When using direct cannulation of the lymphatic vessel, such as in the use of osmotic or insulin pumps to deliver antigen, ultrasound can be used to position the needle directly into the lymphatic vessel and to monitor its positioning over the period of treatment.

The following non-limiting examples are illustrative of the present invention.

EXAMPLES Materials and Methods For Examples 1-5

Mice: The generation of T cell receptor transgenic mice (TCR+ mice) in which approx. 90% of the CD8+ T cells express a TCR recognizing the immunodominant LCMV-glycoprotein epitope (gp-peptide aa33-41, p33:K.AVYNFATC-SEQ ID NO:569) presented on H-2D^(b), has been described in detail. All experimental mice were between 8 and 12 weeks of age and bred and held under strict pathogen free conditions at the Institut Für Labortierkunde at the University of Zurich.

Viruses: LCMV (Armstrong strain) was originally obtained from Dr. M. B. A. Oldstone, Scripps Clinics and Research Foundation, LaJolla, San Diego, Calif. Seed virus was grown on BHK cells and plagued on MC57 cells using an immunological focus assay, as described previously.

Osmotic pump: ALZA model #1007b.

In vivo protection assays for specific CTL activity: The in vivo assay for the detection of CTL activity by challenge infections with LCMV has been described in detail previously (Oehen et al. 1991). Briefly, mice are intravenously challenged with 2×10³ pfu of LCMV (Armstrong), After 4 days the titer of LCMV is determined using the above mentioned immunological focus assay.

Primary ex vivo cytotoxicity against LCMV-gp: Mice were injected intravenously with 10 μg of p33. After 36 hours spleen single cell suspensions were coincubated for 5 h with ⁵¹Cr-labeled syngeneic EL-4 (H-2^(b)) target cells, that were either pulsed with p33 or left unpulsed. Specific lysis was calculated as [(experimental ⁵¹Cr release−spontaneous ⁵¹Cr release)/(total ⁵¹Cr release−spontaneous ⁵¹Cr release)×100%].

LCMV induced foot pad swelling reaction: Mice were infected with LCMV (Armstrong) by intradermal injection into the hind footpad (5000 pfu in 30:1), Footpad thickness was measured daily with a spring loaded caliper. Footpad swelling is calculated as (measured thickness−thickness before injection)/(thickness before injection).

Example 1 Continuous Release of Peptide Antigen Using Osmotic Pump Induces Potent CTL Response in C5BL/6 Mice

C57BL/6 mice were either intravenously injected with a single dose of 50 μg p33 (including 500 ng GM-CSF) (circles) or were implanted with a micro-osmotic pump releasing a mixture of 50 μg of p33 and 500 ng GM-CSF over a time period of 7 days (triangles), or were left naïve (data not shown). After 7 days mice were sacrificed to prepare single cell suspensions from the spleen. Spleen cells were restimulated in vitro for 5 days by p33 pulsed in the presence of low amounts of IL-2. Specific cytotoxicity was measured using ⁵¹Cr-labeled EL-4 target cells pulsed with p33. Specific lysis of EL-4 target cells without p33 was less than 16% for all effectors. The results are shown in FIG. 1.

Example 2 Continuous Release of Antigen Induces CTL Immunity Against Virus in C57BL/6 Mice

C57BL/6 mice were either intravenously injected with a single dose of 50 μg p33 (including 500 ng GM-CSF. Pharmingen) or were implanted with a microsomotic pump releasing a mixture of 50 μg of p33 and 500 ng GM-CSF over a time period of 7 days, or were left naive. After 7 days specific CTL activity was assessed in vivo using anti-viral protection assays. C5713L/6 mice were intravenously challenged with LCMV Armstrong strain (2×10³ p.f.u.). After 4 days mice were sacrificed and LCMV titers were determined in spleens using an immunological focus assay. Mice implanted with osmotic pump showed significantly lower virus titers indicating active CTL immunity against the virus (Table V).

TABLE V C57BL/6 Mice Virus Titer (loglo) Single injection 4.2 Single injection 4.6 Single injection 4.0 Pump delivered 2.2 Pump delivered 1.8 Pump delivered 2.0 Unprimed 4.8 Unprimed 3.8 Unprimed 4.4

Example 3 Continuous Release of Antigen Maintains Potent CTL Effectors in TCR Transgenic Mice

TCR transgenic mice were either intravenously injected with a single dose of 50 μg p33 (circles) or were implanted with a microsomotic pump releasing a mixture of 50 μg of p33 (triangles), or left naïve (squares). After 36 hours mice were sacrificed to prepare single cell suspensions from the spleen which were assayed ex vivo for p33-specific cytotoxicity using ⁵¹Cr-labeled EL-4 target cells pulsed with p33. Similarly mice were either intravenously injected with a single dose of 50 μg p33 (circles) or were implanted with a micro-osmotic pump releasing a mixture of 50 μg of p33 over a time period of 7 days (triangles), or were left naïve (squares). After 7 days mice were sacrificed to prepare single cell suspensions from the spleen to assay ex vivo p33-specific cytotoxicity using ⁵¹Cr-labeled EL-4 target cells pulsed with p33. Specific lysis of EL-4 target cells without p33 was less than 18% for all effectors. The results are shown in FIGS. 2A and 2B.

Continuous Release of Antigen Maintains Protective CTL Response Against Virus Infection.

After 7 days TCR transgenic mice were challenged by intradermal LCMV injection into their hind foot pads (2×10³ pfu in 30 μl). The absence of a foot pad swelling reaction, as observed in mice with an implanted pump (triangles), indicates that at the time point of injection there was active CTL immunity inhibiting local replication of the virus in the foot pad. In contrast, foot pad swelling, as observed in mice injected with the peptide as a single bolus (circles) and naive control mice (data not shown), indicated that LCMV successfully replicated in the foot pad in the absence of protective CTL. The results are shown in FIG. 2C.

Example 4 Direct Delivery of Antigen into Lymphatic Organ Dramatically Increases Efficiency of CTL Induction

TCR transgenic mice were injected with graded doses of gp-peptide p33 either subcutaneously (S.C.), intravenously (I.V.) or directly into the spleen (I.S.) via a small abdominal incision. The efficiency of CTL induction was assessed by measuring gp-specific CTL activity 24 hours after injection. CTL activity is known to peak one day after injection of peptide. Mice were sacrificed to prepare single cell suspensions from draining lymph nodes or from spleen to assay ex vivo p33-specific cytotoxicity using ⁵¹Cr-labeled EL-4 target cells pulsed with p33. Specific lysis of EL-4 target cells without p33 was less than 12% for all effectors. The results are shown in FIG. 3.

Example 5 Dendritic Cells Purified from Mice Receiving Intrasplenic Injection of Peptide Potently Stimulate CTL

The effect of directing peptide delivery into lymphatic system was assessed. Peptide p33 was injected either i.v., s.c. or directly into the spleen of wild-type C57BL/6 mice. After 2 hours, DCs were isolated from the spleen of animals injected either i.s. or i.v., and additionally from the regional draining lymph nodes of animals injected s.c. Cells isolated from these tissues were sorted for DCs using magnetic beads coupled with a monoclonal antibody recognizing the integrin-alpha chain, a marker specific for DCs in spleen and lymph nodes. The positively and the negatively sorted cell fractions were compared regarding their capacity to in vitro stimulate naive CD8+ T cells from TCR transgenic mice specific for LCMV-gp. Only when peptide had been directly injected into the spleen, the DC containing cell fraction stimulated CTL to proliferate, as measured by ³H-thymidine uptake. This indicated that CTL induction after direct injection of peptide into lymphatic organs reflected efficient loading of DCs with peptide. In contrast, the fraction depleted for DC did not induce proliferation and DCs isolated from lymphoid organs of i.v. and s.c injected mice were not effective stimulators. The results are shown in FIG. 4.

While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Example 6 Induction of CTL Response with Naked DNA is Most Efficient by Intra-Lymph Node Immunization

In order to quantitatively compare the CD8⁺ CTL responses induced by different routes of immunization we used a plasmid DNA vaccine (pEGFPL33A) containing a well-characterized immunodominant CTL epitope from the LCMV-glycoprotein (G) (gp33; amino acids 33-41) (Oehen, S., et al. Immunology 99, 163-169 2000), as this system allows a comprehensive assessment of antiviral CTL responses. Groups of 2 C57BL/6 mice were immunized once with titrated doses (200-0.02 μg) of pEGFPL33A DNA or of control plasmid pEGFP-N3, administered i.m. (intramuscular), i.d. (intradermal), i.spl. (intrasplenic), or i.ln. (intra-lymph node). Positive control mice received 500 pfu LCMV i.v. (intravenous). Ten days after immunization spleen cells were isolated and gp33-specific CTL activity was determined after secondary in vitro restimulation. As shown in FIG. 6, i.m. or i.d. immunization induced weakly detectable CTL responses only when high doses of pEFGPL33A DNA (200 μg) were administered. In contrast, potent gp33-specific CTL responses were elicited by immunization with only 2 μg pEFGPL33A DNA i.spl. and with as little as 0.2 μg pEFGPL33A DNA given i.ln. (FIG. 6; symbols represent individual mice and one of three similar experiments is shown). Immunization with the control pEGFP-N3 DNA did not elicit any detectable gp33-specific CTL responses (data not shown).

Example 7 Intra-Lymph Node Immunization is the Most Efficient Way to Induce Antiviral Anamnestic CTL Responses

Similar thresholds for CTL detection were observed when a different readout system was used. Groups of 2 C57BL/6 mice were immunized once with titrated doses of pEFGPL33A DNA (0.2-200 μg) and positive control mice received 500 pfu LCMV i.v., as above. Ten days later they were challenged with 5×10⁴ pfu LCMV i.v. Four days after challenge spleen cells were isolated and ex vivo CTL activity was assayed. This time point is too early to detect any primary CTL response to LCMV infection in naive mice (FIG. 7, Controls), but it allows the detection of anamnestic CTL responses in mice which have been previously immunized (FIG. 7, LCMV). As before, mice immunized with 200 μg intramuscularly showed only weak anamnestic CTL responses following LCMV challenge, which were not detectable when lower immunizing doses of DNA were used (FIG. 7). Those immunized by the i.spl. route showed strong anamnestic CTL responses which titered out at an immunizing dose of 2 μg pEFGPL33A DNA, while the i.ln. route of immunization was again more efficient with anamnestic CTL responses detectable when only 0.2 μg pEFGPL33A DNA was administered (FIG. 7).

These results from examples 6 and 7 clearly demonstrate that administration of plasmid DNA directly into lymphoid tissues is 100- to 1000-fold more efficient than intradermal or intramuscular routes for the induction of CTL responses. In addition, they show that the intra-lymph node route is around 10-fold more efficient than the intrasplenic route.

Example 8 Naked DNA Elicits Superior Protection Against Systemic and Peripheral Virus Infection by Intra-Lymph Node Compared to Intramuscular Immunization

To examine whether the enhanced CTL responses elicited following i.ln. immunization with plasmid DNA were able to qualitatively influence antiviral immunity, we used challenge infections with LCMV or with recombinant vaccinia virus expressing the LCMV-G (Vacc-G2) as models of systemic and peripheral virus infection, respectively. When systemic antiviral immunity was assessed by challenging the immunized mice (groups of 3 C57BL/6 mice) with a high dose of LCMV i.v. (500 pfu), mice which had been immunized once with 200 μg pEGFPL33A DNA i.m. showed only partial and incomplete protection against systemic LCMV challenge, while those which had received 20 μg of pEFGPL33A DNA by the i.spl. or i.ln. routes were completely protected (FIG. 8A).

Eradication of Vacc-G2 infection from peripheral organs such as ovaries, is dependent upon the presence of high levels of recently activated effector CD8+ T cells (Kündig, T. M. et al. Proc. Natl. Acad. Sci. USA 93, 9716-9723, 1996; Bachmann, M. F., et al. Proc. Natl. Acad. Sci. USA 94, 640-645, 1997). Groups of 3 C57BL/6 mice were immunized four times at 6 day intervals with pEFGPL33A DNA administered either i.m. (100 μg per immunization) or i.ln. (10 μg per immunization). Five days after the last immunization they were challenged with 5×10⁶ pfu Vacc-G2 i.p. and vaccinia titers in ovaries were assessed after a further 5 days. Repeated i.m. immunization with pEFGPL33A DNA had no influence on the growth of Vacc-G2 in peripheral tissues (FIG. 8B). In contrast, mice which were repetitively immunized with pEFGPL33A DNA by the i.ln. route were completely protected against peripheral infection with Vacc-G2 (FIG. 8B).

These results illustrate that although repeated i.m. immunization with naked DNA induced detectable CTL responses, these were never of sufficient magnitude to offer protection against virus infection. In contrast, immunization with 10-fold lower amounts of DNA directly into lymphoid organs elicited quantitatively and qualitatively stronger CTL responses, which gave complete protection against systemic or peripheral virus challenge.

Example 9 Intra-Lymph Node DNA Immunization Elicits Anti-Tumor Immunity

To examine whether the potent CTL responses elicited following i.ln. immunization were able to confer protection against peripheral tumors, groups of 6 C57BL/6 mice were immunized three times at 6-day intervals with 10 μg of pEFGPL33A DNA or control pEGFP-N3 DNA. Five days after the last immunization small pieces of solid tumors expressing the gp33 epitope (EL4-33) were transplanted s.c. into both flanks and tumor growth was measured every 3-4d. Although the EL4-33 tumors grew well in mice that had been repetitively immunized with control pEGFP-N3 DNA (FIG. 9), mice which were immunized with pEFGPL33A DNA i.ln. rapidly eradicated the peripheral EL4-33 tumors (FIG. 9).

Example 10 Differences in Lymph Node DNA Content Mirrors Differences in CTL Response Following Intra-Lymph Node and Intramuscular Injection

pEFGPL33A DNA was injected i.ln. or i.m. and plasmid content of the injected or draining lymph node was assessed by real time PCR after 6, 12, 24, 48 hours, and 4 and 30 days. At 6, 12, and 24 hours the plasmid DNA content of the injected lymph nodes was approximately three orders of magnitude greater than that of the draining lymph nodes following i.m. injection. No plasmid DNA was detectable in the draining lymph node at subsequent time points (FIG. 10). This is consonant with the three orders of magnitude greater dose needed using i.m. as compared to i.ln. injections to achieve a similar levels of CTL activity. CD8^(−/−) knockout mice, which do not develop a CTL response to this epitope, were also injected i.ln. showing clearance of DNA from the lymph node is not due to CD8⁺ CTL killing of cells in the lymph node. This observation also supports the conclusion that i.ln. administration will not provoke immunopathological damage to the lymph node.

Example 11 Stability of Plasmid in Different Formulations

DNA is a relatively stable molecule in the kind of formulations of interest to test and thus little loss of material would be noted if the total amount of DNA were to be measured. Instead, the ratio of supercoiled to open-circle DNA was measured. Since a single nick anywhere in either strand of the DNA molecule will allow a supercoiled plasmid to relax to an open circle conformation this is an exquisitely sensitive indication of damage to the DNA backbone. Plasmid was formulated, placed in vials in triplicate and incubated at 37° C. After 1, 3 and 7 days aliquots were removed, subjected to anion exchange HPLC, and the peak areas corresponding to supercoiled and open-circle DNA compared (see FIG. 11). Nine formulations were tested:

10% Ethanol, 0.25 mM EDTA, Citrate Phosphate pH 7.6

10% Ethanol, 0.25 mM EDTA, Citrate Phosphate pH 7.4

1% Ethanol, 0.5 mM EDTA, Citrate Phosphate pH 7.4

1% Ethanol, 0.5 mM EDTA, 1×PBS pH 7.4

0.5% Benzyl Alcohol, 0.25 mM EDTA, Citrate Phosphate pH 7.6

1% Benzyl Alcohol, 1% Ethanol, 0.5 mM EDTA, Citrate Phosphate pH 7.6

1% Benzyl Alcohol, 1% Ethanol, 0.5 mM EDTA, 0.1M TRIS pH 7.4

1% Benzyl Alcohol, 1% Ethanol, 0.5 mM EDTA, 0.1M TRIS pH 8.2

1% Benzyl Alcohol, 1×PBS pH 8.2

Citrate Phosphate Buffer pH. 7.4 was made by mixing 9.15 parts (by volume) of 0.1M citric acid with 90.85 parts of 0.2M Sodium Phosphate Dibasic. Citrate Phosphate Buffer pH. 7.6 was made by mixing 6.35 parts (by volume) of 0.1M citric acid with 93.65 parts of 0.2M Sodium Phosphate Dibasic. These solutions were then added to the other components to create a 2× buffer which was mixed with a equal volume of DNA in water. Thus the final concentrations of citrate and phosphate in the above buffers was on the order of 3 mM and 90 mM, respectively.

Formulations 1-3 and 6 gave superior results (see FIG. 11).

Example 12 Stability of Formulated Plasmid in Operating MINIMED 407C Infusion Pumps

Using a modification (final concentrations of 0.1 M sodium phosphate dibasic and 0.05 M citric acid; pH 7.6±0.2) of formulation 6 above, aliquots of 80, 160, and 320 μg DNA/ml were prepared and loaded in triplicate into MINIMED 3.0 reservoir syringes. A 200 μl sample was dispensed and the reservoir syringes were inserted into MINIMED 407C infusion pumps and assembled with SILHOUETTE infusion sets fitted with 3.1 mm catheters. The pump assemblies, set to dispense 10 μl/hour and with the catheters inserted into collection vials, were placed in 37° C. incubators. At 4 and 8 days the catheters were briefly detached and 200 μl bolus samples dispensed directly from the reservoir. The concentration of supercoiled DNA was determined for each sample by anion exchange HPLC and the use of a standard curve constructed with known concentrations of DNA. Plotting the resultant concentrations versus time allows one to derive a slope indicating the rate of loss of supercoiled DNA. The average (of the triplicate samples) rates of loss were −0.056±1.88, 0.24±1.01, and 0.048±0.49 μg DNA/day for the 80, 160, and 320 μg DNA/ml samples, respectively. None of these differ significantly from zero.

Example 13 Administration of a DNA Plasmid Formulation of a Therapeutic Vaccine for Melanoma to Humans

SYNCHROTOPE TA2M, a melanoma vaccine encoding HLA-A2-restricted tyrosinase epitopes was formulated in 1% Benzyl alcohol, 1% ethyl alcohol, 0.5 mM EDTA, citrate-phosphate, 7.6. Aliquots of 80, 160, and 320 μg DNA/ml were prepared for loading into MINIMED 407C infusion pumps. The catheter of a SILHOUETTE infusion set is placed into an inguinal lymph node visualized by ultrasound imaging. The assembly of pump and infusion set was originally designed for the delivery of insulin to diabetics and the usual 17 mm catheter has been substituted with a 31 mm catheter for this application. The infusion set is kept patent for 4 days (approximately 96 hours) with an infusion rate of about 25 μl/hour resulting in a total infused volume of approximately 2.4 ml. Thus the total administered dose per infusion will be approximately 200, 400, and 800 μg, respectively, for the three concentrations described above. Following an infusion subjects will be given a 10 day rest period before starting a subsequent infusion. Given the continued residency of plasmid DNA in the lymph node after administration (as in example 10) and the usual kinetics of CTL response following disappearance of antigen, this schedule will be sufficient to maintain the immunologic CTL response. 

1. A method of inducing or sustaining a CTL response in a mammal, wherein the method comprises: delivering an antigen to the mammal at a level sufficient to induce a CTL response to the antigen in the mammal, wherein the antigen is delivered to the mammal by pumping a physiologically-acceptable acellular composition of the antigen from a device held external to the mammal's body positioned to deliver the antigen-containing composition so that the antigen reaches the mammal's lymph system; and maintaining the antigen in the mammal's lymphatic system over time sufficient to induce or maintain the CTL response specific to the antigen.
 2. The method of claim 1, wherein said pumping a physiologically-acceptable composition of the antigen comprises pumping from a device held external to the mammal's body through a transmission line.
 3. The method of claim 2, wherein said transmission line comprises a catheter.
 4. The method of claim 3, wherein said catheter is at least 20 mm in length.
 5. The method of claim 1, wherein the antigen is delivered to an area of high lymphatic drainage in the mammal.
 6. The method of claim 1, wherein said maintaining step comprises maintaining the antigen in the mammal's lymphatic system sufficient to maintain the CTL response for a period of time that is substantially co-extensive with a desired duration of the CTL response.
 7. The method of claim 1, wherein delivery of the antigen is directly to the lymphatic system.
 8. The method of claim 7, wherein said delivery of the antigen directly to the lymphatic system comprises delivery to an inguinal or axillary lymph node.
 9. The method of claim 1, wherein said delivering comprises delivering the antigen in the form of a vector comprising a nucleic acid encoding the antigen.
 10. The method of claim 1, wherein said delivering comprises delivering the antigen in the form of a peptide.
 11. The method of claim 1, wherein said antigen is provided as a component of a microorganism or mammalian cell.
 12. The method of claim 1, wherein said antigen is a disease-matched antigen.
 13. The method of claim 12, wherein said disease is cancer.
 14. The method of claim 13, wherein said antigen is a tumor-associated antigen.
 15. The method of claim 12, wherein said disease is an infectious disease.
 16. The method of claim 15, wherein said antigen is a microbial antigen.
 17. The method of claim 1, wherein said antigen is a non-peptide antigen.
 18. The method of claim 1, wherein said level is sufficient to induce an increase in a pre-existing response.
 19. A method of sustaining a CTL response in a mammal, wherein the method comprises: delivering an antigen to the mammal at a level sufficient to sustain a CTL response to the antigen in the mammal, wherein the antigen is delivered to the mammal by pumping a physiologically-acceptable acellular composition of the antigen from a device held external to the mammal's body positioned to deliver the antigen-containing composition so that the antigen reaches the mammal's lymph system; and maintaining the antigen in the mammal's lymphatic system over time sufficient to sustain the CTL response specific to the antigen. 